Cell surface display of polypeptide isoforms by stop codon readthrough

ABSTRACT

The present invention inter alia pertains to a method for generating or selecting a eukaryotic host cell expressing a desired level of a polypeptide of interest, comprising:
         a) providing a plurality of eukaryotic host cells comprising a heterologous nucleic acid comprising at least one cassette (Cas-POI) comprising at least a first polynucleotide (Pn-POI) encoding the polypeptide of interest, at least one stop codon downstream of the first polynucleotide, and a second polynucleotide downstream of the stop codon encoding an immunoglobulin transmembrane anchor or a functional variant thereof;   b) cultivating the eukaryotic host cells to allow expression of the polypeptide of interest such that at least a portion of the polypeptide of interest is expressed as a fusion polypeptide comprising the immunoglobulin transmembrane anchor or a functional variant thereof, wherein said fusion polypeptide is being displayed on the surface of said host cell;   c) selecting at least one eukaryotic host cell based upon the presence or amount of the fusion polypeptide displayed on the cell surface.       

     Also provided are host cells, comprising respectively designed heterologous nucleic acids and methods for producing a polypeptide using respective host cells.

The present invention pertains to a method for selecting high-producingmammalian host cells as well as to vectors and host cells suitable foruse in a respective method. Furthermore, the present invention pertainsto a method for efficiently producing polypeptides with a high yield.

Selection of high-producing cell lines is an important first step in thedevelopment of any bio-process and is one of the greatest challenges inbiotechnology. One problem is that such high-producing clones are rare,may spend much of their energy on polypeptide production, and thus havereduced growth rates. This leads to overgrowth and non- or lowproducing-cells. However, in the production of polypeptides it isdesirable to obtain cell lines producing the polypeptide of interestwith a high yield. Traditionally, high-producing cell lines wereselected by rounds of limiting dilution cloning followed by productanalysis. However, this traditional route has several draw-backs as itis both labor intensive and costly. Beyond that, the whole process istime consuming and can take several months to complete and even thenthere is no guarantee that the clone cell line will be stable and souseful for industrial bioprocessing. Furthermore, selection of thehighest producers can be compromised by practical limitations on thenumber of cells that can be screened thereby potentially reducing theefficiency of selection of low abundance, high productivity cells.

Therefore, there have been many efforts to provide alternative methodsfor selecting high producing clones. For example, flow cytometry hasmade it easier to monitor productivity and to isolate cells withspecific characteristics. Important advantages of flow cytometry includethe ability to screen large numbers of cells rapidly, with thecapability to distinguish cell sub-populations and the ability toefficiently select low abundance cells demonstrating the desiredcharacteristics. Most of the traditional approaches for selecting highproductivity cells utilizing flow cytometry were established for theselection of hybridoma cells.

One approach is based on the cell surface antibody content of hybridomacells displaying an increased amount of cell surface antibodies that canbe identified and recovered through the use of fluorescence labeledantibodies. However, a quantitative correlation has not been broadlydocumented.

Further approaches were developed to select cells based on secretedantibody as an alternative strategy to circumvent some of thelimitations of cells surface antibody selection. One approach applies anaffinity matrix; the other uses a gel microdroplet technology. Theformer method is based on creation of an artificial affinity matrix,specific for the secreted product of interest. Secreted molecules bindto the affinity matrix on the surface of the secreting cell and aresubsequently labeled with specific fluorescent reagents for flowcytometric analysis and cell sorting.

Microdroplet encapsulation involves complete encapsulation of singlecells in agarose beads. These beads contain specific capture antibodiesand so simultaneously capture secreted product and prevent cross feedingof product between cells.

Other methods rely on the co-expression of marker genes, which aredetectable by flow cytometry. Disadvantages are a weak linkage ofexpression of the marker gene (for example green fluorescence protein)to the expression of the gene of interest. Furthermore, the expressionof the marker gene costs cells additional energy and might inducestress.

An alternative method relies on an inducible co-expression of membranebound capturing proteins. The membrane bound capturing proteins areanchored to the cell surface and capture the secreted polypeptide assoon as they are released from the cells. Those captured molecules canthen be detected on the surface of the cell. However, geneticallyengineered host cells are necessary and also cross-feeding ofnon-producing cells might occur.

Also secreted products transiently associated to the cell membrane havebeen used in order to select producing cells. However, cross feeding ofnon-producing cells occurs and this method has a rather high backgroundactivity. Furthermore, it was found that it is not possible to performseveral rounds of enrichment and selection.

Therefore, there is a need in developing a technology for selectinghigh-producing host cells. It is thus the object of the presentinvention to provide a method for detecting high producing recombinanthost cells within a large populating of non-, low-, and/ormedium-producing cells and to provide a method for producingpolypeptides with a high yield.

The present invention solves this problem by providing a method forenriching or selecting at least one eukaryotic host cell expressing adesired level of a polypeptide of interest, comprising:

-   -   a) providing a plurality of eukaryotic host cells comprising a        heterologous nucleic acid comprising at least one cassette        (Cas-POI) comprising at least a first polynucleotide (Pn-POI)        encoding the polypeptide of interest, at least one stop codon        down-stream of the first polynucleotide, and a second        polynucleotide downstream of the stop codon encoding an        immunoglobulin transmembrane anchor or a functional variant        thereof;    -   b) cultivating the eukaryotic host cells to allow expression of        the polypeptide of interest such that at least a portion of the        polypeptide of interest is expressed as a fusion polypeptide        comprising the immunoglobulin transmembrane anchor or a        functional variant thereof, wherein said fusion polypeptide is        being displayed on the surface of said host cell;    -   c) selecting at least one eukaryotic host cell based upon the        presence or amount of the fusion polypeptide displayed on the        cell surface.

A “heterologous nucleic acid” refers to a polynucleotide sequence thathas been introduced into a host cell e.g. by the use of recombinanttechniques such as transfection. The host cell may or may not comprisean endogenous polynucleotide corresponding to, respectively beingidentical to the heterologous polynucleotide. However, in particular theterm “heterologous nucleic acid” refers to a foreign polynucleotideintroduced into the host cell. Introduction may be achieved e.g. bytransfecting a suitable vector that may integrate into the genome of thehost cell (stable transfection). In case the heterologous nucleic acidis not inserted into the genome, the heterologous nucleic acid can belost at the later stage e.g. when the cells undergo mitosis (transienttransfection). Both variants are suitable, however, stable transfectionis preferred. Suitable vectors might also be maintained in the host cellwithout integrating into the genome, e.g. by episomal replication.However, also other techniques are known in the prior art forintroducing a heterologous nucleic acid into a host cell which are alsodescribed in further detail below.

A “polynucleotide” is a polymer of nucleotides which are usually linkedfrom one deoxyribose or ribose to another and refers to DNA as well asRNA, depending on the context. The term “polynucleotide” does notcomprise any size restrictions.

A “cassette” describes a group of polynucleotide elements, operablylinked to each other, comprising e.g. a polynucleotide (Pn-POI) encodingthe polypeptide of interest, a polynucleotide encoding a immunoglobulintransmembrane anchor or a functional variant thereof, a polynucleotideencoding a marker, regulatory elements and/or other polynucleotidesdescribed herein. A “cassette” as used herein comprises at least twopolynucleotide elements. A cassette may or may not comprise regulatoryelements as polynucleotides such as e.g. a promoter, an enhancer and/ora polyA site. According to one embodiment, the cassette is an“expression cassette” suitable for expressing a polypeptide. Anexpression cassette comprises at least one transcription initiationelement, e.g. a promoter, as regulatory element operably linked to acoding region, e.g. a polynucleotide (Pn-POI) encoding a polypeptide ofinterest, which is then accordingly under the transcriptional control ofsaid transcription initiation element. An expression cassette may alsocomprise suitable regulatory elements for transcription termination,such as e.g. a polyA site.

The “cassette (Cas-POI)” comprises at least a first polynucleotide(Pn-POI) encoding the polypeptide of interest, at least one stop codondownstream of the first polynucleotide, and a second polynucleotidedownstream of the stop codon encoding an immunoglobulin trans-membraneanchor or a functional variant thereof. Preferably, the cassette(Cas-POI) is an expression cassette (Exp-POI).

The “expression cassette (Exp-POI)” defines an expression cassettesuitable for expressing a polypeptide of interest (POI). As expressioncassette it comprises at least one transcription initiation element.Said expression cassette (Exp-POI) either comprises the polynucleotide(Pn-POI) encoding the polypeptide of interest as part of the codingregion or comprises a site suitable for inserting a respectivepolynucleotide (Pn-POI) encoding the polypeptide of interest—dependingon the used embodiment of the present invention which are described infurther detail below.

The general concept of the present invention is to place a stop codonbetween the polynucleotide (Pn-POI) encoding the polypeptide of interestand the polynucleotide encoding the immunoglobulin transmembrane anchoror a functional variant thereof which allows anchoring of thepolypeptide to the cell surface. The term “immunoglobulin transmembraneanchor” and “immunoglobulin transmembrane domain” are used as synonymsherein. The stop codon constitutes or is part of a translationtermination signal and may be the natural stop codon of thepolynucleotide encoding the polynucleotide of interest and thus the stopcodon that is naturally used to terminate translation. The design of thecassette (Cas-POI) results upon expression in the generation of twodifferent polypeptides when the first and second polynucleotides aretranscribed into a transcript, which is optionally processed, andsubsequently translated. According to one translation variant,translation of the transcript is aborted at the (at least one) stopcodon located between the polynucleotide (Pn-POI) encoding thepolypeptide of interest and the polynucleotide encoding animmunoglobulin transmembrane anchor or a functional variant thereof. Thetermination of the translation at said stop codon results in apolypeptide product—not comprising the transmembrane anchor. Accordingto the second translation variant, translation reads through said atleast one stop codon, thereby rendering a translation product comprisingthe polypeptide of interest and fused thereto the immunoglobulintransmembrane anchor or a functional variant thereof which is capable ofanchoring the fusion polypeptide to the cell membrane. Such fusionpolypeptide is transferred and fixed to the cell surface via thecomprised immunoglobulin transmembrane anchor or functional fragmentthereof. It is an important feature of the present invention that uponexpression of the cassette (Cas-POI) the termination of the translationat said in frame stop codon is to a certain extent “leaky”, astranslational read-through occurs, thereby rendering the describedfusion polypeptide. As this translational read-through occurs at adefined proportion—which can also be influenced by the choice and numberof the stop codon(s) and the regions adjacent to the stop codon, inparticular the nucleotide following the stop codon as well as by theculture conditions—the level of surface bound fusion polypeptidedirectly correlates with the expression level of the polypeptide ofinterest. The amount of fusion polypeptide present on the cell surfaceis thus to a certain extent proportional to the overall expression levelof the polypeptide of interest by the respective cell, as there is astrong linkage between the surface expression of the fusion polypeptideand the productivity of the host cell in expressing the polypeptide ofinterest. The level of surface bound fusion polypeptide is thereforerepresentative for the overall productivity of the individual cell andallows the selection of at least one eukaryotic host cell based upon thepresence or amount of the fusion polypeptide displayed on the cellsurface. A selection cycle comprising the steps a), b) and c) allows theefficient and reproducible identification and isolation of highproducing eukaryotic host cells.

Suitable detection/selection methods like immunostaining, flowcytometry, fluorescent microscopy, MACS, affinity based methods such asmagnetic beads and similar techniques allow the identification,selection and/or enrichment of high-producing cells based on thepresence and level of surface bound fusion polypeptide. Therefore, thepresent invention leads to a drastic reduction in screening efforts byallowing the selection and also the enrichment of at least onehigh-producing cell or a population of high producing cells from apopulation of non-, low- and/or medium producing cells. It is alsopossible to perform several rounds of selection and/or enrichment,preferably two or three. E.g. a sufficient or even high expressingeukaryotic host cell or population of host cells may be selected byusing a detection compound such as e.g. an antibody or fragment thereofrecognizing the membrane anchored fusion polypeptide. Said detectioncompound may carry a label and can thus be detected by common detectionmethods.

According to the teachings of the present invention, at least a fragmentof an immunoglobulin transmembrane anchor/domain is used in order toanchor the polypeptide of interest to the cell surface. In case afragment instead of a full-length immunoglobulin transmembrane anchor isused, the respective fragment shall allow anchoring of the fusionpolypeptide to the cell surface. The immunoglobulin transmembraneanchor/domain or functional fragment thereof is embedded in and therebytightly anchored to the cell membrane. This tight anchoragedistinguishes the anchors of the present invention from e.g. a GPIanchor. The immunoglobulin transmembrane anchor used according to thepresent invention provides a very robust and thus durable anchoring ofthe fusion polypeptide to the cell surface which is also not or at leastless susceptible to proteolytic shedding. This is confirmed by theperformed product analysis after purification. No immunoglobulintransmembrane anchor (or immunoglobulin transmembrane anchor fragment)containing heavy chain species are found in the performed massspectrometry analysis. This is an important advantage over the prior artas also the risk of contaminations of the secreted soluble polypeptideof interest by shedded fusion polypeptides is reduced. Furthermore,according to the analysed characteristics of the expressed polypeptidesof interest, also no significant differences to material fromconventional clones/expression systems are found.

Cells obtained by the method of the present invention have a higheraverage expression level than cells cloned by limited dilution orsimilar methods. They also have a higher average expression level thancells cloned for example by flow cytometry after transfection of astandard vector not comprising the specific transmembrane domain/anchoraccording to the present invention.

Cells that are identified as a result of the screening/selectionprocedure of the present invention will generally be isolated and may beenriched from non-selected cells of the original cell population. Theycan be isolated and cultured as individual cells. They can also be usedin one or more additional rounds of selection, optionally for additionalqualitative or quantitative analysis, or can be used e.g. in developmentof a cell line for protein production. According to one embodiment, anenriched population of high producing cells selected as described aboveis directly used as population for the production of the polypeptide ofinterest with high yield.

Advantageously, the observed growth behaviour and productivity of clonesco-expressing the transmembrane variant of the polypeptide of interest,in particular antibodies and clones derived from a classical vectorsetup not co-expressing the transmembrane variant of the polypeptide ofinterest appear to be the same. Furthermore, also the clonal productionstability appears to be equally good.

Also provided is a method for producing a polypeptide of interest withhigh yield, the method comprising:

-   -   a) providing a plurality of eukaryotic host cells comprising a        heterologous nucleic acid comprising at least one cassette        (Cas-POI) comprising a first polynucleotide (Pn-POI) encoding        the polypeptide of interest, at least one stop codon downstream        of the first polynucleotide, and a second polynucleotide        downstream of the stop codon encoding an immunoglobulin        transmembrane anchor or a functional variant thereof;    -   b) cultivating the eukaryotic host cells to allow expression of        the polypeptide of interest such that at least a portion of the        polypeptide of interest is expressed as a fusion polypeptide        comprising the immunoglobulin transmembrane anchor or a        functional variant thereof, wherein said fusion polypeptide is        being displayed on the surface of said host cell;    -   c) selecting at least one eukaryotic host cell based upon to the        presence or amount of the fusion polypeptide displayed on the        cell surface;    -   d) culturing the selected eukaryotic host cell in culture medium        under conditions that allow for expression of the polypeptide of        interest.

The expressed polypeptide of interest may be obtained by disrupting thehost cells. The polypeptides may also be expressed, e.g. secreted intothe culture medium and can be obtained therefrom. Also combinations ofthe respective method are possible. Thereby, polypeptides can beproduced and obtained/isolated efficiently with high yield. The obtainedpolypeptides may also be subject to further processing steps such ase.g. purification and/or modification steps in order to produce thepolypeptide of interest in the desired quality. According to oneembodiment, said host cells are cultured under serum-free conditions. Asis outlined above, by inserting at least one stop codon between thepolynucleotide (Pn-POI) encoding the polypeptide of interest and thesecond polynucleotide encoding an immunoglobulin transmembrane anchor orfunctional fragment thereof, the selection of high expressing host cellsis possible, thereby allowing the production of the polypeptide ofinterest with high yield. The selection/enrichment step of the presentinvention is thus an integral and important component of the overallproduction process.

The use of an immunoglobulin transmembrane anchor or functional fragmentthereof according to the teachings of the present invention isparticularly advantageous when producing immunoglobulin molecules, assaid immunoglobulin transmembrane anchor is naturally suitable to fiximmunoglobulin molecules to the cell surface. Surprisingly, it is foundthat the immunoglobulin transmembrane anchor can be used when expressingimmunoglobulin molecules in mammalian host cells such as CHO cells. Thisis surprising, as the prior art assumed that the co-expression of the Igalpha and Ig beta receptor chains is necessary in said cells in order toachieve surface—and accordingly cell membrane anchored—expression ofantibodies when using the Ig transmembrane domain as anchor. Theseco-receptors are e.g. naturally expressed in B-cells and B-cellderivatives such as hybridoma or myeloma cells (e.g. SP2/0 cells) butare not expected to be expressed in non B-cells such as CHO cells.However, it is found that despite the missing expression of theco-receptors the surface display of the polypeptides of interest and inparticular of immunoglobulin molecules worked well on non B-cellderivatives such as CHO cells when using the Ig transmembraneanchor/domain. Hence, according to one embodiment a eukaryotic host cellis used which is not a B-cell or a B-cell derivative. Accordingly, aeukaryotic, preferably a mammalian host cell used which does notnaturally express the Ig alpha and Ig beta receptor chains. Thus,preferably, the host cell is a CHO cell. Furthermore, according to oneembodiment, no artificial co-expression of the Ig alpha and Ig betareceptor chain occurs in said eukaryotic host cell.

Any immunoglobulin transmembrane anchor or functional fragment thereofcan be used according to the teachings of the present invention. Inparticular, the immunoglobulin trans-membrane anchor is selected fromthe group consisting of immunoglobulin transmembrane anchors derivedfrom IgM, IgA, IgE, IgG and/or IgD or functional variants thereof.Preferably, the immunoglobulin transmembrane anchor is derived fromIgG1, IgG2, IgG3 and/or IgG4. Particularly suitable is an IgG1immunoglobulin transmembrane anchor or a functional variant thereof.Preferred examples of an IgG derived transmembrane anchor are shown inSEQ ID NO: 2 and SEQ ID NO: 7.

According to one embodiment, the immunoglobulin transmembrane anchorcomprises a cytoplasmatic domain. The use of an immunoglobulintransmembrane anchor comprising a cytoplasmatic domain is preferred asit provides a very tight anchorage of the fusion polypeptide to the cellsurface. Particularly suitable is the use of an immunoglobulincytoplasmatic domain. According to one embodiment, the immunoglobulincytoplasmatic domain is derived from IgG, IgA and IgE or functionalvariants of the foregoing. These immunoglobulin cytoplasmatic domainsare larger than the ones derived from IgD and IgM. SEQ ID NO: 4 and SEQID NO: 6 show suitable amino acid sequences of IgG derived cytoplasmaticdomains that can be used as cytoplasmatic domain. A preferred example ofan IgG derived trans-membrane anchor which comprises an IgG derivedcytoplasmatic domain is shown in SEQ ID NO: 3.

Thus, the immunoglobulin transmembrane anchor may comprise a polypeptidesequence as shown in SEQ ID NO: 2 and/or SEQ ID NO: 3 or functionalvariants, in particular functional fragments thereof, which allow theanchoring of the fusion polypeptide to the surface of the host cell.

The nucleotide sequence of a section of a suitable cassette (Cas-POI) isshown as SEQ ID NO:1. The shown detail comprises an in frame stop codonsuitable for translational read through and a polynucleotide encoding aparticularly suitable immunoglobulin (Ig) transmembrane domain that canbe used according to the teachings of the present invention. The stopcodon is located in frame downstream of the polynucleotide encoding thepolypeptide of interest and thus the polynucleotide sequence that istranscribed and processed into an amino acid sequence. The codingsequence refers to the sequence that is translated into amino acids.Thus, the stop codon does not belong to the coding sequence andaccordingly to the polynucleotide encoding the polypeptide of interest.The stop codon can be the natural stop codon of the polynucleotideencoding the polypeptide of interest. In this case, no additional stopcodon(s) need(s) to be but may be present (see above).

The second polynucleotide of the cassette (Cas-POI) may encode animmunoglobulin trans-membrane anchor or a functional variant thereof,which comprises a polypeptide sequence shown as SEQ ID NO: 2. SEQ ID NO:3 shows a further variant of a suitable immunoglobulin transmembranedomain, also comprising a cytoplasmatic domain (the cytoplasmatic domainalone is also shown as SEQ ID NO: 4), the putative amino acidscorresponding to the leaky stop codon and the additional codon (WL) anda connecting region (the connecting region alone is also shown as SEQ IDNO: 5). Also other amino acids can be present in the positioncorresponding to the at least one stop codon and the adjacent codon,depending on the chosen stop codon and/or number of stop codons and theused adjacent codon(s). As these amino acids are only present in thefusion polypeptide, they do not alter the amino acid sequence of thepolypeptide of interest. Accordingly, an immunoglobulin transmembranedomain comprising a polypeptide sequence as shown as SEQ ID NO: 2 or 3or a functional variant, in particular a functional fragment thereof canbe used as a transmembrane anchor according to the teachings of thepresent invention and hence in the described methods, as well as in thedescribed vectors and host cells.

“A functional variant” of an immunoglobulin transmembrane anchoraccording to the present invention include immunoglobulin transmembraneanchors having one or more amino acid sequence exchanges (e.g.deletions, substitutions or additions) with respect to the amino acidsequence of the respective natural immunoglobulin transmembrane domainand functional fragments of the foregoing, which allow transmembraneanchoring of the fusion polypeptide to the cell surface.

As translation termination signal and thus stop codon any one of thethree stop codons that signal termination of protein synthesis (TAA(UAA), TAG (UAG) and TGA (UGA)—also in various tetranucleotide contexts,see below) can be used between the polynucleotide (Pn-POI) encoding thepolypeptide of interest and the polynucleotide encoding theimmunoglobulin transmembrane anchor or a functional variant thereof,depending on the desired level of suppression (read-through). As isoutlined above, the stop codon may also be the natural stop codon of thepolynucleotide encoding the polypeptide of interest. Preferably, saidtranslation termination signal has an incomplete termination efficiencyin order to promote translational read through. The “leakyness” of thestop codon is also influenced by the codon(s) adjacent and thusdownstream of the at least one stop codon, in particular the firstnucleotide may influence the transcriptional read-through (see below).

The cassette (Cas-POI) needs to be transcribed in order to allow theexpression of the polypeptide of interest. According to one embodiment,the cassette (Cas-POI) is therefore an expression cassette. According toa further embodiment, the cassette (Cas-POI) is integrated into thegenome of the host cell such that the cassette (Cas-POI) is under thetranscriptional control of a transcription initiation element of thehost cell, such as an promoter.

Transcription of the nucleic acid comprised in the cassette (Cas-POI)results in a transcript comprising at least

-   -   a first polynucleotide, wherein translation of said first        polynucleotide results in the polypeptide of interest;    -   at least one stop codon downstream of said first polynucleotide;    -   a second polynucleotide downstream of said stop codon, wherein        translation of said second polynucleotide results in the        immunoglobulin transmembrane anchor or a functional variant        thereof.

At least a portion of the transcript is translated into a fusionpolypeptide comprising the polypeptide of interest and theimmunoglobulin transmembrane anchor or a functional variant thereof bytranslational read-through of the at least one stop codon. Translationalread-through may occur naturally due to the choice of the stopcodon/design of the translation termination signal or can be induced byadapting the culturing conditions, e.g. by using a terminationsuppression agent (see below).

The cassette (Cas-POI) used in the method of the invention may compriseonly a single stop codon upstream of the coding sequence for theimmunoglobulin transmembrane anchor or fragment thereof. However, it isalso possible to use a series of two or more stop codons, e.g. two orthree, or four stop codons, which may be the same or different. Also thecontext of the stop codon, i.e. the trinucleotide stop codon itself aswell as the nucleotide(s) respectively codon immediately downstream ofthe stop codon, has an influence on the read-through levels. However, itneeds to be ensured that a certain level of translational read-throughstill occurs in order to allow the production of the fusion polypeptidewhich may be achieved according to one embodiment by adjusting theculture conditions.

The primary transcript may be a pre-mRNA comprising introns. Arespective pre-mRNA would be processed (spliced) into mRNA.Alternatively, transcription may result directly in mRNA. Duringtranslation of the mRNA transcript there is usually a natural level ofbackground read-through of the stop codon(s) or a respectiveread-through level can be induced by adapting the culture conditions.This read-through level results in a certain proportion of fusionpolypeptides that also depends on the number and nature of the used stopcodon(s), the downstream stop codon and in particular thetetranucleotide context of the stop codon(s) and the culture conditions.Accordingly, a certain proportion of fusion polypeptide is producedaccording to the teachings of the present invention despite the presenceof the stop codon. These fusion polypeptides comprise the immunoglobulintransmembrane anchor or a functional variant thereof, tightly anchoringthe fusion polypeptides to the cell surface. As a result, the fusionpolypeptides are displayed at the surface of the host cells, and cellsdisplaying high levels of membrane-anchored recombinant fusionpolypeptides (indicating a high level of secreted polypeptide) can beselected e.g. by flow cytometry, in particular by fluorescence activatedcell sorting (FACS) when contacted with an appropriately labelleddetection compound.

Suitable translation termination signals and thus stop codons and stopcodon settings with incomplete translation termination efficiency can bedesigned as described in the prior art (see e.g. Li et al. 1993, Journalof Virology 67 (8), 5062-5067; McCughan et al. 1995 Proc. Natl. Acad.Sci. 92, 5431-5435; Brown et al 1990, Nucleic Acids Research 18 (21)6339-6345, herein incorporated by reference).

According to one embodiment, the following stop codon setting is used;the stop codon is shown in bold and underlined:

-   TGACTA nucleotide sequence of the stop coding setting on the coding    strand and thus on the DNA level; the stop codon is shown in bold    and underlined-   UGACUA nucleotide sequence of the stop codon setting on the RNA    level-   W L putative amino acids corresponding to the stop codon and the    adjacent codon if translational read-through occurs; shown is thus    the most likely read-through product of the shown polynucleotide

The additional amino acids that are incorporated into the fusionpolypeptide due to the read-through of the stop codon can be of any kindas long as the fusion protein is displayed on the cell surface. As saidadditional amino acids are only incorporated into the fusionpolypeptide, the amino acid of the polypeptide of interest remainsunaltered.

In addition to the possible use of multiple stop codons following thepolynucleotide (Pn-POI) encoding the polypeptide of interest, it willnormally be advantageous to use multiple stop codons downstream of thesequence encoding the immunoglobulin transmembrane anchor or functionalfragment thereof. The use of multiple stop codons in this position, e.g.up to about ten stop codons, such up to about six or eight stop codons,such as about two, three, four or five stop codons, will ensureefficient termination of translation.

The amount of fusion polypeptide present and thus detectable on the cellsurface usually increases during polypeptide synthesis as the fusionpolypeptide remains anchored to the cell membrane and thus accumulateson the cell surface as expression continues. According to oneembodiment, the cassette (Cas-POI) is constructed such that stop codonread-through results in approximately ≦50%, ≦25%, ≦15%, ≦10%, ≦5%,≦2.5%, ≦1.5%, ≦1% or less than ≦0.5% fusion polypeptide. The remainingportion is produced as the polypeptide form not comprising theimmunoglobulin transmembrane anchor or functional fragment thereof. Asdescribed, the level of stop codon read through can be influenced by thechoice and number of the stop codon(s) and the regions adjacent to thestop codon, in particular the nucleotide following the stop codon, aswell as by the culture conditions used during step b).

Depending on factors such as the natural level of backgroundread-through for a given stop codon in a given construct, it may in somecases be desirable to use more than one stop codon between thepolynucleotide (Pn-POI) encoding the polypeptide of interest and thepolynucleotide encoding the immunoglobulin transmembrane anchor in orderto further reduce background read-through levels (see above). Thegeneral advantage of a rather low read-through level is a higherstringency in the subsequent selection/enrichment and sorting procedure,which is preferably done by FACS, leading to a better resolution of highproducing versus ultra high producing clones. If read-through levels aretoo high, saturation of the cell surface capacity for membrane boundpolypeptides might occur, which may prevent discrimination of expressionlevels, in particular of high expression levels. Therefore, a rather lowread-through level is advantageous in order to select ultra highexpressing clones. Accordingly, preferably only ≦5%, ≦2% or even ≦1.5%of the transcript is translated into a fusion polypeptide.

However, it is also possible to increase the read-through level ifnecessary/desired, e.g. by using a termination suppression agent duringculturing. The use of a termination suppression agent in the culturemedia during step b) is one way of influencing the level of stop codonread through by the culture conditions. A termination suppression agentis a chemical agent which is able to suppress translational terminationresulting from the presence of a stop codon. In particular, thetermination suppression agent is an antibiotic belonging to theaminoglycoside group. Aminoglycoside antibiotics are known for theirability to allow insertion of alternative amino acids at the site of astop codon, thereby resulting in “read-through” of a stop codon or stopcodon setting that otherwise normally would result in translationtermination. Aminoglycoside antibiotics include G-418, gentamycin,paromomycin, hygromycin, amikacin, kanamycin, neomycin, netilmicin,streptomycin and tobramycin. However, as a low read-through level isadvantageous, selection is preferably performed in the absence of atermination suppression agent.

The present invention is applicable to any type of host cell in whichtranslational stop codon read-through occurs at least to a smallpercentage or can be induced by the addition of a terminationsuppression agent. Examples of suitable eukaryotic host cells aremammalian hosts cells which include e.g. Chinese hamster ovary (CHO)cell lines, green monkey cell lines (COS), mouse cells (for exampleNS/0), baby hamster kidney (BHK) cell lines and human cells and celllines. Preferably, the host cell is a CHO cell line.

While one selection cycle is sufficient to identify good producing hostcells, according to one embodiment, two or more selection cycles areperformed, wherein in each selection cycle at least one eukaryotic hostcell is selected based upon the presence or amount of the fusionpolypeptide displayed on the cell surface. The experimental resultsdemonstrate that a second selection cycle usually leads to improvedresults.

According to one embodiment, the selection step c) comprises contactingthe plurality of host cells with a detection compound binding the fusionpolypeptide and selecting at least one host cell based upon the presenceor amount of the detection compound bound to the cell surface.

The detection compound used for binding to the fusion polypeptide mayhave at least one of the following characteristics:

-   -   said compound is labelled;    -   said compound is fluorescently labelled;    -   said compound is an antigen;    -   said compound is an immunoglobulin molecule or a binding        fragment thereof;    -   said compound is protein-A, -G, and/or -L.

The detection compound used for binding the fusion polypeptide at thecell surface can for example be an immunoglobulin molecule or a fragmentthereof such as an antibody or antibody fragment, recognising the fusionpolypeptide. Basically all accessible portions of the fusion polypeptidecan be detected, thereunder also the portion corresponding to thepolypeptide of interest which is secreted in parallel to the fusionpolypeptide in soluble form.

According to one embodiment, the detection compound is an antigen. Thisembodiment is suitable, if the expressed polypeptide of interest is forexample immunoglobulin molecule or a fragment thereof such as anantibody, binding the respective antigen.

In order to allow detection and selection, said detection compound usedfor binding the fusion polypeptide may be labelled. The labelleddetection compound that binds the fusion polypeptide displayed on thecell surface thereby labels respectively stains the cell surface. Thehigher the amount of fusion polypeptide that is expressed by the hostcell, the more labelled detection compound is bound. This has theadvantage that the selection of the host cells can be easily performedas not only the presence but also the amount of the bound detectioncompound can be determined due to the label. To select high producinghost cells, those host cells are selected from the population of hostcell which are most effectively respectively intensively labelled by thedetection compound. A fluorescent label is preferred as this allows easydetection by fluorescence detecting methods such as for example flowcytometry. Suitable fluorescent labels are known to the skilled person.

According to one embodiment, one or more selection cycles, preferablytwo or three, may be performed to select at least one eukaryotic hostcell based on the degree of binding of the detection compound to thecell surface. According to this embodiment, at least one eukaryotic hostcell is selected in each selection cycle based upon the amount of bounddetection compound. Thus, those host cells that were mosteffectively/intensively labelled are selected based upon the degreerespectively amount of cell surface staining. E.g. the top 5% or the top2% of the host cells can be selected.

In case several eukaryotic host cells are supposed to be selectedtogether as a pool (so called pool enrichment), several cells, e.g. atleast 10, at least 50, at least 500, at least 1000 or at least 50.000are selected and included in a cell pool. This embodiment isparticularly advantageous for quickly obtaining larger amounts of thepolypeptide of interest as the cell pool comprising several highproducing host cells selected according to the teachings of the presentinvention can be expanded more quickly than e.g. a cell clone.

Thus, besides the application for selective cell cloning, the presentinvention can also be used for pool enrichment of high-producing cellswhereby titers comparable to clonal cell lines can be achieved.

High-producing host cells can be isolated and/or a population of highproducing cells can be enriched based on the degree of binding of thedetection compound to the cell surface, in particular the fusionpolypeptide. Binding of the detection compound to the fusion polypeptideon the surface of the host cell can be detected by flow cytometry,preferably fluorescence activated cell sorting (FACS).

In a preferred embodiment, host cells comprising a high amount of fusionpolypeptides which accordingly depict a high signal are sorted usingfluorescence-activated cell sorting (FACS). In the context of thepresent invention, FACS sorting is particularly advantageous, since itallows rapid screening of large numbers of host cells to identify andenrich those cells which express the polypeptide of interest with a highyield. As according to the preferred embodiment approximately only 5% orless of the polypeptide are produced as a fusion polypeptide, a higherfluorescence detected on the cell surface would correspond to a higherexpression also of the polypeptide of interest, which can be e.g.secreted into the culture medium. Those cells, showing the highestfluorescence rate can be identified and isolated by FACS. A positive andstatistically significant correlation between fluorescence, asdetermined by FACS and the amount of produced polypeptide is found andconfirmed by the examples. Therefore, FACS sorting can be used not onlyfor a qualitative analysis to identify cells expressing a polypeptide ofinterest in general, but can actually be used quantitatively to identifythose host cells that express high levels of the polypeptide ofinterest. Therefore, high-producing host cells can be selected/enrichedbased on the degree of binding of the labelled detection compound to thefusion polypeptide, which is anchored to the cell surface. Thereby thebest producing cells can be selected/enriched. The experimental resultsshow that using the selection procedure according to the presentinvention in combination with FACS analysis led to a significantreduction of non-producing clones in the selected cell populations.Furthermore, the highly increased average productivity of the clonesallows the drastic reduction of clone screening efforts e.g. in the cellline development process for biopharmaceutical production. Thus, celllines for a much higher number of candidates or projects can bedeveloped with less resources compared to classical screeningapproaches. Also, this process allows the evaluation of the productivitypotential and clonal distribution of transfected and selected pools bysurface staining and FACS analysis instead of time consumingproductivity assays. Surface staining may also be used to analyze theclonal production stability with regards to the homogeneity of the cellpopulation. Non- or low-producing sub-populations that may arise wouldbe easily detectable.

According to one embodiment, the cassette (Cas-POI) and/or (Cas-POI′)further comprises

-   -   a polynucleotide (Pn-TAG) encoding an affinity tag located        downstream of the at least one stop codon which is located        downstream of the first polynucleotide and wherein said        polynucleotide (Pn-TAG) is located upstream of the second        polynucleotide encoding an immunoglobulin transmembrane anchor        or a functional variant thereof and/or    -   a polynucleotide (Pn-MARKER) encoding a selectable marker.

To provide the polynucleotide (Pn-TAG) as defined above between the stopcodon and the polynucleotide encoding the immunoglobulin transmembraneanchor has the advantage that an affinity tag is incorporated into thefusion protein. As the affinity tag is located downstream of the atleast one stop codon, it is only included in the fusion variant of thepolypeptide of interest. An “affinity tag” refers to a short amino acidsequence which can be detected/bound by binding compounds/agents such asantibodies. Basically, the affinity tag serves as a target for captureagents and/or detection compounds. As it is located between thepolypeptide of interest and the immunoglobulin transmembrane anchor or afunctional variant thereof, it is also displayed on the cell surface andis accordingly accessible e.g. for detection compounds. The affinity tagmay thus also function as target for the detection compound in order toallow the selection of suitable eukaryotic host cells. To use e.g. awell characterised affinity tag as target for detection/selection isadvantageous as existing and well characterised detection compounds maybe used for detection. Furthermore, the same detection compound can beused for different kinds of polypeptides of interest to be expressed.The generation of detection compounds specific for the differentpolypeptide of interest would be obsolete according to this embodimentas the same detection compound specific for the affinity tag could beused. As the affinity tag constitutes an integral part of the fusionpolypeptide it is also tightly anchored to the surface of the eukaryotichost cell due to the presence of the transmembrane anchor. The tightlyanchored fusion polypeptides should not be susceptible to shedding (seeabove).

Shedding of membrane bound fusion proteins may constitute—depending onthe intended use of the secreted polypeptide—a contamination problemeven if shedding is a rare event when using an immunoglobulintransmembrane anchor. E.g. when expressing therapeuticpolypeptides/proteins, it is desirable to obtain the secreted product aspure as possible. When using an affinity tag such as e.g. a His tag,said affinity tag would be at least partially comprised in the sheddedprotein. Due to the presence of the affinity tag it is possible toremove shedded fusion polypeptides (if present) from the sample ofsecreted polypeptides by using conventional affinity purificationprocedures (e.g. Ni-NTA in case of a His tag). The affinity tag istherefore useful in order to easily remove potential contaminations fromthe sample.

Furthermore, the affinity tag can be used in order to control the purityof the expressed/obtained polypeptide. For applications where highlypure proteins/polypeptides are needed it may also beadvantageous/mandatory to provide suitable assays to demonstrate thatthe product obtained is pure and accordingly does not comprisecontaminations due to shedded fusion polypeptides. Such an assay couldbe based on the detection of the affinity tag. As the affinity tag isonly present in the fusion polypeptide, it may serve as a specificmarker for the presence of fusion polypeptides (or degraded/sheddedversions thereof) in the sample. If the affinity tag can still bedetected in the obtained product when using a detection compoundspecific for the affinity tag, there are still traces of shedded fusionpolypeptide in the sample and the sample may need—depending on theamount—further purification. If no affinity tag can be detected in thesample, no or respectively very low amounts of shedded fusionpolypeptide should be present in the analysed sample thereby ensuringthat the sample is sufficiently pure for the intended application.

Accordingly, when producing the polypeptide of interest, the obtainedpolypeptide of interest can be further processed by

-   -   removing contaminations of shedded fusion polypeptide by        affinity purification targeting the affinity tag and/or    -   detecting the presence or absence of shedded fusion protein by        targeting the affinity tag.

Suitable examples for affinity tags are e.g. V5, a H is Tag, FLAG,Strep, HA, c-Myc or the like. Suitable affinity tags can also beartificially created.

According to a further embodiment, the cassette (Cas-POI) and/or(Cas-POI′) comprises a further polynucleotide (Pn-MARKER) encoding aselectable marker. Preferably, said selectable marker is locateddownstream of the polynucleotide encoding the immunoglobulintransmembrane anchor and is thus upon expression of the constructlocated on the cytoplasmatic site of the cell membrane when the fusionpolypeptide is displayed. According to one embodiment, no stop codon islocated between the polynucleotide encoding the immunoglobulintransmembrane anchor/domain or a functional variant thereof and thepolynucleotide (Pn-MARKER), as they are supposed to be expressed as afusion. Suitable stop codons and transcription termination signalsshould be provided downstream of the coding sequence of thepolynucleotide (Pn-MARKER) to ensure efficient transcription andtranslation termination after expression of the polynucleotide(Pn-MARKER). Said polynucleotide (Pn-MARKER) can e.g. be a drugresistance gene or a reporter gene. Suitable examples are describedherein. According to one embodiment, green fluorescence protein (GFP) orluciferase is used as reporter. This allows the selection of theeukaryotic host cells based upon two fusion protein characteristics.

According to one embodiment, the cassette (Cas-POI) and/or (Cas-POI′) isan expression cassette. Persons skilled in the art will be capable ofselecting suitable vectors, expression control sequences and hosts forperforming the methods of the invention. For example, in selecting avector, the host must be considered because the vector may need to beable to replicate in it and/or be able to integrate into the chromosome.Suitable vectors that can be used in the selection and productionmethods according to the present invention are also described below andin the claims.

Also a vector nucleic acid suitable for expressing at least onepolypeptide of interest in a eukaryotic, preferably a mammalian hostcell is provided, comprising at least one cassette (Cas-POI) comprisingan insertion site for a first polynucleotide (Pn-POI) encoding thepolypeptide of interest and/or a first polynucleotide (Pn-POI) encodinga polypeptide of interest, at least one stop codon downstream of thefirst polynucleotide, and a second polynucleotide downstream of the stopcodon encoding an immunoglobulin transmembrane anchor or a functionalvariant thereof.

A “vector nucleic acid” according to the present invention is apolynucleotide capable of carrying at least one foreign nucleic acidfragment. A vector nucleic acid functions like a “molecular carrier”,delivering fragments of nucleic acids into a host cell. It may compriseat least one expression cassette comprising regulatory sequences.Preferably, the vector nucleic acid comprises at least one expressioncassette. Foreign polynucleotides may be inserted into the expressioncassette(s) of the vector nucleic acid in order to be expressedtherefrom. The vector nucleic acid according to the present inventionmay be present in circular or linearized form. The term “vector nucleicacid” also comprises artificial chromosomes or similar respectivepolynucleotides allowing the transfer of foreign nucleic acid fragments.

A respective vector can be used as expression vector in order to performthe screening and production methods described above. The advantages ofa respective vector nucleic acid are also described above in conjunctionwith the screening method.

Said vector nucleic acid may further comprise at least

-   -   a first polynucleotide (Pn-POI) encoding the polypeptide of        interest;    -   an expression cassette (Exp-MSM) comprising a mammalian        selectable marker gene; and/or    -   an expression cassette (Exp-MASM) comprising a mammalian        amplifiable, selectable marker gene.

The expression cassette (Exp-MSM) defines the expression cassettecomprising a mammalian selectable marker gene.

The expression cassette (Exp-MASM) defines the expression cassettecomprising a mammalian amplifiable, selectable marker gene.

The terms “5′” and “3′” is a convention used to describe features of anucleic acid sequence related to either the position of genetic elementsand/or the direction of events (5′ to 3′), such as e.g. transcription byRNA polymerase or translation by the ribosome which proceeds in 5′ to 3′direction. Synonyms are upstream (5′) and downstream (3′).Conventionally, DNA sequences, gene maps, vector cards and RNA sequencesare drawn with 5′ to 3′ from left to right or the 5′ to 3′ direction isindicated with arrows, wherein the arrowhead points in the 3′ direction.Accordingly, 5′ (upstream) indicates genetic elements positioned towardsthe left hand side, and 3′ (downstream) indicates genetic elementspositioned towards the right hand side, when following this convention.

The arrangement and orientation of the expression cassettes is also animportant aspect. According to one embodiment, the expression cassette(Exp-MASM) is located 5′ and the expression cassette (Exp-MSM) islocated 3′ of the expression cassette (Exp-POI). Further expressioncassettes may be inserted between the expression cassettes (Exp-POI) and(Exp-MSM), such as e.g. an additional expression cassette (Exp-POI′) forexpressing an additional polypeptide of interest (described in furtherdetail below). The expression cassettes (Exp-MASM), (Exp-POI) and(Exp-MSM) are preferably all arranged in the same 5′ to 3′ orientation.The inventors found, that this particular vector nucleic acidconfiguration allows the fast generation of high yielding cell lines.

According to one alternative, the expression cassette (Exp-POI) does notcomprise the polynucleotide (Pn-POI) encoding the polypeptide ofinterest. Thus, an “empty” expression vector with an expression cassette(Exp-POI) is provided which does not yet comprise the polynucleotide(Pn-POI) encoding the polypeptide of interest. However, saidpolynucleotide (Pn-POI) encoding the polypeptide of interest can beincorporated into the expression cassette (Exp-POI) by using appropriatecloning methods, for example by using restriction enzymes in order toinsert the polynucleotide (Pn-POI) encoding the polypeptide of interestinto the expression cassette (Exp-POI). For this purpose the expressioncassette (Exp-POI) may comprise e.g. a multiple cloning site (MCS) whichcan e.g. be used in all reading frames. A respective “empty” vectornucleic acid can e.g. be provided to customers, which then insert theirspecific polynucleotide of interest into the expression cassette(Exp-POI). The polynucleotide (Pn-POI) encoding the polypeptide ofinterest is inserted such that a stop codon is present between thepolynucleotide (Pn-POI) encoding the polypeptide of interest and thepolynucleotide encoding the transmembrane anchor or a functional variantthereof. The expression cassette (Exp-POI) may also comprise areplacement polynucleotide or a stuffer nucleic acid sequence, which canbe excised and replaced by the polynucleotide (Pn-POI) encoding thepolypeptide of interest. The present invention also provides a vectornucleic acid as described above, comprising an expression cassette(Exp-POI) comprising a first polynucleotide (Pn-POI) encoding thepolypeptide of interest, at least one stop codon down-stream of thefirst polynucleotide, and a second polynucleotide downstream of the stopcodon encoding an immunoglobulin transmembrane anchor or a functionalvariant thereof. This embodiment pertains basically to the finalexpression vector nucleic acid. Basically the same applies in case acassette (Cas-POI) is used instead of an expression cassette (Exp-POI).

According to one embodiment, the vector nucleic acid is circular and theexpression cassette (Exp-MSM) is arranged 3′ of the expression cassette(Exp-POI) and the expression cassette (Exp-MASM) is arranged 3′ of theexpression cassette (Exp-MSM).

The expression vector according to the present invention may comprise anadditional expression cassette (Exp-POI′) for expressing a polypeptideof interest. In the final vector nucleic acid, said additionalexpression cassette (Exp-POI′) comprises the additional polynucleotidefor expressing the additional polypeptide of interest. Depending on thepolypeptides to be expressed, said additional expression cassette(Exp-POI′) may or may not comprise a polynucleotide encoding a membraneanchor (or a signal peptide for attaching a respective anchor, such as aGPI anchor), which is separated from the polynucleotide encoding theadditional polypeptide of interest by a stop codon. Therefore, it isalso possible that several expression cassettes for expressing differentpolypeptides are arranged in the expression vector according to thepresent invention. However, only the expression cassette (Exp-POI) needsto have the leaky stop codon assembly and thus a stop codon downstreamof the first polynucleotide (Pn-POI) encoding the polypeptide ofinterest and a second polynucleotide downstream of the stop codonencoding an immunoglobulin transmembrane anchor or a functional variantthereof, the additional expression cassettes (Exp-POI′) may or may nothave a respective stop codon assembly.

A respective embodiment using at least two expression cassettes(Exp-POI) and (Exp-POI′) for expressing the polypeptides of interest isparticularly advantageous, in case an immunoglobulin molecule orfunctional fragment thereof is expressed. Accordingly, a vector nucleicacid for expressing at least one immunoglobulin molecule or a functionalfragment thereof is provided, comprising

-   -   an expression cassette (Exp-POI) comprising a first        polynucleotide encoding the heavy and/or the light chain of the        immunoglobulin molecule or a functional fragment thereof, at        least one stop codon downstream of the first polynucleotide, and        a second polynucleotide downstream of the stop codon encoding at        least an immunoglobulin transmembrane anchor or functional        fragment thereof; and/or    -   an additional expression cassette (Exp-POI′) comprising a        polynucleotide encoding the corresponding light and/or the heavy        chain of an immunoglobulin molecule or a functional fragment        thereof. The expression cassette (Exp-POI′) encodes the        immunoglobulin chain that corresponds to the immunoglobulin        chain of the expression cassette (Exp-POI) (i.e. if the        expression cassette (Exp-POI) encodes the heavy chain, the        expression cassette (Exp-POI′) encodes the light chain and vice        versa). Thus, a functional immunoglobulin molecule (or fragment        thereof) can be expressed from the vector.

It is preferred that the heavy chain or a functional fragment thereof isexpressed from the expression cassette (Exp-POI) and is thus accordingto a certain extent expressed as a fusion polypeptide. The correspondinglight chain or a functional fragment thereof is according to oneembodiment expressed from an expression cassette (Exp-POI′). Saidexpression cassette (Exp-POI′) may be located on the same vector nucleicacid. However, it may also be located on a separate vector nucleic acid.However, it is preferred that the expression cassettes (Exp-POI) and(Exp-POI′) are located on one vector nucleic acid. It is also possibleto express both chains (the heavy chain and the corresponding lightchain) from one expression cassette. E.g. they may be expressed as afusion polypeptide comprising a self-splicing signal or a proteasesusceptible site in order to obtain two separate chains. Also possibleis a bi- or multicistronic set up, wherein two or more polypeptides (POIand POI′) are obtained from one mRNA which may comprise e.g. one or moreinternal ribosomal entry sites.

According to one embodiment, a vector nucleic acid for expressing atleast one immunoglobulin molecule or a functional fragment thereof isprovided, comprising

-   -   an expression cassette (Exp-POI) comprising a first        polynucleotide encoding the heavy chain of an immunoglobulin        molecule or a functional fragment thereof, at least one stop        codon downstream of the first polynucleotide, and a second        polynucleotide downstream of the stop codon encoding an        immunoglobulin transmembrane anchor or a functional variant        thereof; and    -   an additional expression cassette (Exp-POI′) comprising a        polynucleotide encoding the corresponding light chain of an        immunoglobulin molecule or a functional fragment thereof.

Preferably, the expression cassette (Exp-POI) comprises the heavy chainand both expression cassettes (Exp-POI) and (Exp-POI′) are arranged inthe same orientation. Preferably, the expression cassette (Exp-POI′) isarranged 5′ of the expression cassette (Exp-POI). To arrange theexpression cassette for the light chain 5′ to the expression cassette ofthe heavy chain proved to be beneficial regarding the expression rate ofimmunoglobulin molecules. According to one embodiment, it is alsodestined to design the expression vector such, that the expressioncassette(s) already comprise the immunoglobulin transmembrane anchor andthe at least one leaky stop codon (e.g. comprised in a stuffer sequence)and, optionally, at least part of the constant regions of animmunoglobulin molecule. The fragments encoding the variable parts ofthe immunoglobulin molecules can then be inserted by the user/customerinto the expression cassettes by using appropriate cloning strategies inorder to obtain the final expression vector.

Non-limiting examples for mammalian selectable marker genes that can becomprised in the expression cassette (Exp-MSM) include antibioticresistance genes e.g. conferring resistance to G418; hygromycin (hyg orhph, commercially available from Life Technologies, Inc. Gaithesboro,Md.); neomycin (neo, commercially available from Life Technologies, Inc.Gaithesboro, Md.); zeocin (Sh Ble, commercially available fromPharmingen, San Diego Calif.); puromycin (pac,puromycin-N-acetyl-transferase, available from Clontech, Palo AltoCalif.), ouabain (oua, available from Pharmingen) and blasticidin(available from Invitrogen). Said mammalian selectable marker genesallow the selection of mammalian host cells comprising said genes andthus of host cells comprising the vector. The term “gene” as used hereinnot only refers to the coding sequence of the wildtype gene but alsorefers to a nucleic acid sequence encoding a functional variant of theselectable marker providing the intended resistance. Hence, alsotruncated or mutated versions of a wild type gene are encompassed aslong as they provide the intended resistance. The mammalian selectablemarker gene preferably comprises foreign regulatory elements such ase.g. a strong constitutive promoter. According to a preferredembodiment, said expression cassette (Exp-MSM) comprises a gene encodingan enzymatically functional neomycin phosphotransferase (I or II) whichpreferably comprises foreign regulatory elements such as e.g. a strongconstitutive promoter such as the SV40 promoter. This embodiment workswell in combination with the use of a gene encoding an enzymaticallyfunctional DHFR as a mammalian amplifiable selectable marker gene.

Mammalian, amplifiable selectable marker genes incorporated in theexpression cassette (Exp-MASM) allow selection of vector-containing hostcells as well gene amplification. A non-limiting example for a mammalianamplifiable, selectable marker gene is the dihydrofolate reductase(DHFR) gene encoding the DHFR enzyme. The mammalian, amplifiableselectable marker gene preferably comprises foreign regulatory elementssuch as e.g. a strong constitutive promoter. Other systems currently inuse are among others the glutamine synthetase (gs) system (Bebbington etal., 1992) and the histidinol driven selection system (Hartmann andMulligan, 1988). These amplifiable markers are also selectable markersand can thus be used to select those cells that obtained the vector.DHFR and glutamine synthetase provide good results. In both casesselection occurs in the absence of the appropriate metabolite(hypoxanthine and thymidine in case of DHFR, glutamine in the case ofGS), preventing growth of non-transformed cells. With amplifiablesystems such as the DHFR system, expression of a recombinant protein canbe increased by exposing the cells to certain agents promoting geneamplification such as e.g. methotrexate (MTX) in case of the DHFRsystem. E.g. the coding sequence of the wildtype DHFR gene or a DHFRmutant allowing e.g. a selection of dhfr+ cell lines may be used. Asuitable inhibitor for GS promoting gene amplification is methioninesulphoximine (MSX). Exposure to MSX also results in gene amplification.

According to one embodiment, said expression cassette (Exp-MASM)comprises a gene encoding an enzymatically functional dihydrofolatereductase (DHFR) which is preferably used in conjunction with the SV40promoter.

Accordingly, vector nucleic acids are provided wherein the expressioncassettes comprise at least one promoter and/or promoter/enhancerelement. Although the physical boundaries between these two controlelements are not always clear, the term “promoter” usually refers to asite on the nucleic acid molecule to which an RNA polymerase and/or anyassociated factors binds and at which transcription is initiated.Enhancers potentiate promoter activity, temporally as well as spatially.Many promoters are transcriptionally active in a wide range of celltypes. Promoters can be divided in two classes, those that functionconstitutively and those that are regulated by induction orderepression. Promoters used for high-level production of proteins inmammalian cells should be strong and preferably active in a wide rangeof cell types. Strong constitutive promoters which drive expression inmany cell types include but are not limited to the adenovirus major latepromoter, the human cytomegalovirus immediate early promoter, the SV40and Rous Sarcoma virus promoter, and the murine 3-phosphoglyceratekinase promoter, EF1a. Good results are achieved with the expressionvector of the present invention when the promoter and/or enhancer iseither obtained from CMV and/or SV40.

According to one embodiment, the expression cassette(s) for expressingthe polypeptide(s) of interest comprise(s) a stronger promoter and/orenhancer than the expression cassettes for expressing the selectablemarkers. This arrangement has the effect that more transcript for thepolypeptide of interest is generated than for the selection markers. Itis advantageous that the production of the polypeptide of interest whichis secreted is dominant over the production of the selection markers,since the individual cell capacity for producing heterologous proteinsis not unlimited and should thus be focused to the polypeptide ofinterest.

According to one embodiment, the expression cassettes (Exp-POI) and(Exp-POI′) (if present) which is/are used for expressing the polypeptideof interest comprise a CMV promoter/enhancer as regulatory elements. Theexpression cassettes (Exp-MSM) and (Exp-MASM), which preferably expressthe DHFR and the neomycin marker genes, comprise an SV40 promoter or aSV40 promoter/enhancer. The CMV promoter is known to be one of thestrongest promoters available for mammalian expression and leads to avery good expression rate. It is considered to give significantly moretranscript than the SV40 promoter.

Most eukaryotic nascent mRNAs possess a poly A tail at their 3′ endwhich is added during a complex process that involves cleavage of theprimary transcript and a coupled polyadenylation reaction. The polyAtail is advantageous for mRNA stability and transferability. Hence, theexpression cassettes of the vector according to the present inventionusually comprise a polyadenylation site. There are several efficientpolyA signals that can be used in mammalian expression vectors,including those derived from bovine growth hormone (bgh), mousebeta-globin, the SV40 early transcription unit and the Herpes simplexvirus thymidine kinase gene. However, also synthetic polyadenylationsites are known (see e.g. the pCl-neo expression vector of Promega whichis based on Levitt et al, 1989, Genes Dev. 3, (7): 1019-1025). Thepolyadenylation site can be selected from the group consisting ofSV40polyA site, such as the SV40 late and early poly-A site (see e.g.plasmid pSV2-DHFR as described in Subramani et al, 1981, Mol. Cell.Biol. 854-864), a synthetic polyA site (see e.g. the pCl-neo expressionvector of Promega which is based on Levitt et al, 1989, Genes Dev. 3,(7): 1019-1025) and a bgh polyA site (bovine growth hormone).

Furthermore, the expression cassettes may comprise an appropriatetranscription termination site. This, as continued transcription from anupstream promoter through a second transcription unit may inhibit thefunction of the downstream promoter, a phenomenon known as promoterocclusion or transcriptional interference. This event has been describedin both prokaryotes and eukaryotes. The proper placement oftranscriptional termination signals between two transcription units canprevent promoter occlusion. Transcription termination sites are wellcharacterized and their incorporation in expression vectors has beenshown to have multiple beneficial effects on gene expression.

The expression cassettes may comprise an enhancer (see above) and/or anintron. According to one embodiment, the expression cassette(s) forexpressing the polypeptide of interest comprise an intron. Most genesfrom higher eukaryotes contain introns which are removed during RNAprocessing. Genomic constructs are expressed more efficiently intransgenic systems than identical constructs lacking introns. Usually,introns are placed at the 5′ end of the open reading frame. Accordingly,an intron may be comprised in the expression cassette(s) for expressingthe polypeptide(s) of interest in order to increase the expression rate.Said intron may be located between the promoter and or promoter/enhancerelement(s) and the 5′ end of the open reading frame of the polypeptideto be expressed. Hence, a vector nucleic acid is provided, wherein atleast the expression cassette (Exp-POI) comprises an intron which isarranged between the promoter and the start codon of the polynucleotidefor expressing the polypeptide of interest. Several suitable introns areknown in the state of the art that can be used in conjunction with thepresent invention.

According to one embodiment, the intron used in the expression cassettesfor expressing the polypeptides of interest, is a synthetic intron suchas the SIS or the RK intron. The RK intron is a strong synthetic intronwhich is preferably placed before the ATG start codon of the gene ofinterest. The RK intron consists of the intron donor splice site of theCMV promoter and the acceptor splice site of the mouse IgG Heavy chainvariable region (see e.g. Eaton et al., 1986, Biochemistry 25,8343-8347, Neuberger et al., 1983, EMBO J. 2(8), 1373-1378; it can beobtained from the pRK-5 vector (BD PharMingen)).

Surprisingly, the placement of an intron at the 3′ end of the openreading frame of the DHFR gene has advantageous effects on theexpression/amplification rate of the construct. The intron used in theDHFR expression cassette is leading to a smaller, non functional variantof the DHFR gene (Grillari et al., 2001, J. Biotechnol. 87, 59-65).Thereby the expression level of the DHFR gene is lowered. This leads toincreased sensitivity for MTX and more stringent selection conditions.Accordingly, a vector nucleic acid is provided, wherein the expressioncassette (MASM) comprises an intron which is located 3′ of theamplifiable selectable marker gene. A suitable intron may be obtainedfrom the pSV2-DHFR vector (see e.g. above).

Said vector may comprise at least one additional expression cassette(Exp-PSM) comprising a prokaryotic selectable marker gene. Saidexpression cassette (Exp-PSM) can be located between the expressioncassettes (Exp-MSM) and (Exp-MASM). Said selectable marker may provide aresistance to antibiotics such as e.g. ampicillin, kanamycin,tetracycline and/or chloramphenicol. Said expression cassette (Exp-PSM)is preferably arranged in the same 5′ to 3′ orientation as the otherexpression cassettes (Exp-POI), (Exp-MSM) and (Exp-MASM).

According to one embodiment, the expression cassette (Exp-POI) and/or(Exp-POI′) comprised in the vector further comprise(s)

-   -   a polynucleotide (Pn-TAG) encoding an affinity tag located        downstream of the at least one stop codon which is located        downstream of the first polynucleotide and wherein said        polynucleotide (Pn-TAG) is located upstream of the second        polynucleotide encoding an immunoglobulin transmembrane anchor        and/or    -   a polynucleotide (Pn-MARKER) encoding a selectable marker.

The advantages are outlined above.

The vector nucleic acid can be transfected into the host cell in itscircular form. Supercoiled vector molecules usually will be convertedinto linear molecules within the nucleus due to the activity of endo-and exonucleases. However, linearization of the vector nucleic acidbefore transfection often improves the efficiency of a stabletransfection. This also as the point of linearization may be controlledif the vector is linearized prior to transfection.

Hence, according to one embodiment of the present invention theexpression vector comprises a predefined restriction site, which can beused for linearization of the vector nucleic acid prior to transfection.Intelligent placement of said linearization restriction site isimportant, because said restriction site determines where the vectornucleic acid is opened/linearized and thus determines theorder/arrangement of the expression cassettes when the construct isintegrated into the genome of the eukaryotic, in particular mammaliancell.

Accordingly, the vector nucleic acid may comprise a linearizationrestriction site for linearizing the vector, wherein said linearizationrestriction site is located between the expression cassettes (Exp-MSM)and (Exp-MASM). Preferably, said linearization restriction site isunique and is only once present in the expression vector nucleic acid.E.g. a linearization restriction site can be used that is recognized bya restriction enzyme having a low cutting frequency in order topatronize that the vector is only cleaved at the linearizationrestriction site but not (or only rarely) e.g. within the expressioncassette(s) or the vector backbone. This can e.g. be encouraged byproviding a restriction site for a restriction enzyme having arecognition sequence of more than six base pairs or which recognizessequences that are under-represented in chromosomal DNA. A suitableexample is the Swal enzyme and the vector may therefore incorporate aSwal recognition site as unique linearization restriction site. In casesaid linearization restriction site is present more than once in thevector nucleic acid sequence (including the polynucleotides encoding thepolypeptide of interest), or in case a restriction enzyme is used whichcuts several times in the vector nucleic acid sequence, it is alsowithin the scope of the present invention to e.g. alter/mutate therestriction sites besides the linearization restriction site which islocated between the expression cassettes (Exp-MSM) and (Exp-MASM), inorder to eliminate those additional restriction sites and to obtain aunique or at least rare linearization restriction site.

In case the vector is used as a standard expression vector intended e.g.as a tool for the expression of several different polypeptides, it isadvantageous to provide a linearization restriction site comprisingmultiple recognition sites for enzymes having a low cutting frequency.The restriction enzymes chosen for linearization should preferably notcut within the expression cassettes for the selectable markers or othervector backbone sequences in order to ensure that the enzyme cuts onlyonce for proper linearization of the vector. By providing alinearization restriction site comprising multiple recognition sites forrestriction enzymes having a low cutting frequency, the user may chose asuitable restriction enzyme for linearization from the provided optionsin order to securely avoid restriction within the polynucleotide(Pn-POI) encoding the polypeptide of interest. However, as is outlinedabove, additional restriction sites may be mutated or a partialrestriction digest could be performed.

Placing the linearization restriction site between the expressioncassette (Exp-MSM) and the expression cassette (Exp-MASM) has the effectthat the expression cassette (Exp-POI) (and further expression cassettesfor expressing the polypeptides of interest—if present) is flanked 5′ bythe expression cassette (Exp-MASM). The expression cassette (Exp-MSM) islocated 3′ of the expression cassette (Exp-POI) upon linearization.Thereby, the expression cassettes (MSM) and (MASM) are separated uponlinearization of the circular vector nucleic acid. If an expressioncassette (Exp-PSM) for a bacterial selection marker is present (seebelow), the linearization restriction site is preferably placed betweenthe expression cassettes (Exp-PSM) and (Exp-MASM). This has the effectthat the bacterial selection marker gene is 3′ and thus “outside” of the“mammalian” parts of the linearized vector nucleic acid. Thisarrangement is favorable since bacterial genes are presumably notadvantageous for mammalian expression as bacterial sequences may lead toincreased methylation or other silencing effects in the mammalian cells.

The polypeptide of interest is not limited to any particular protein orgroup of proteins, but may on the contrary be any protein, of any size,function or origin, which one desires to select and/or express by themethods described herein. Accordingly, several different polypeptides ofinterest may be expressed/produced. The term polypeptide refers to amolecule comprising a polymer of amino acids linked together by apeptide bond(s). Polypeptides include polypeptides of any length,including proteins (e.g. having more than 50 amino acids) and peptides(e.g. 2-49 amino acids). Polypeptides include proteins and/or peptidesof any activity or bioactivity, including e.g. bioactive polypeptidessuch as enzymatic proteins or peptides (e.g. proteases, kinases,phosphatases), receptor proteins or peptides, transporter proteins orpeptides, bactericidal and/or endotoxin-binding proteins, structuralproteins or peptides, immune polypeptides, toxins, antibiotics,hormones, growth factors, vaccines or the like. Said polypeptide may beselected from the group consisting of peptide hormones, interleukins,tissue plasminogen activators, cytokines, immunoglobulins, in particularantibodies or antibody fragments or variants thereof.

As used herein, an “immunoglobulin molecule” as an example for apolypeptide of interest refers to a protein comprising one or morepolypeptides substantially or partially encoded by immunoglobulin genesor fragments of immunoglobulin genes, e.g., a fragment containing one ormore complementarity determining region (CDR). The recognizedimmunoglobulin genes include the kappa, lambda, alpha, gamma, delta,epsilon and mu constant region genes, as well as myriad immunoglobulinvariable region genes. Light chains are typically classified e.g. aseither kappa or lambda.

Heavy chains are typically classified e.g. as gamma, mu, alpha, delta,or epsilon, which in turn define the immunoglobulin classes, IgG, IgM,IgA, IgD and IgE, respectively. Said immunoglobulin can be of anyisotype. Very often IgG (e.g. IgG1) molecules are produced/needed astherapeutic proteins. A typical immunoglobulin (antibody) structuralunit comprises a tetramer. In nature, each tetramer is composed of twoidentical pairs of polypeptide chains, each pair having one “light”(about 25 kD) and one “heavy” chain (about 50-70 kD). The N-terminus ofeach chain defines a variable region of about 100 to 110 or more aminoacids primarily responsible for antigen recognition. The terms variablelight chain (VL) and variable heavy chain (VH) refer to these light andheavy chains respectively.

Antibodies exist as intact immunoglobulins or as a number of wellcharacterized fragments which can e.g. be produced by digestion withvarious peptidases. An antibody fragment is any fragment of an antibodycomprising at least 20 amino acids from said whole antibody, preferablyat least 100 amino acids which at least still has an antigen bindingcapacity. The antibody fragment may comprise the binding region of theantibody such as a Fab fragment, a F(ab)2 fragment, multibodiescomprising multiple binding domains such as diabodies, triabodies ortetrabodies, single domain antibodies or affibodies. An antibody variantis a derivative of an antibody or antibody fragment having the samebinding function but e.g. an altered amino acid sequence. Said antibodyand/or antibody fragment may comprise a murine light chain, human lightchain, humanized light chain, human heavy chain and/or murine heavychain as well as active fragments or derivatives thereof. Hence, it canbe e.g. murine, humane, chimeric or humanized. While various antibodyfragments are defined in terms of the digestion of an intact antibody,one of skill will appreciate that such Fab′ or F(ab)2 fragments may besynthesized de novo either chemically or by utilizing recombinant DNAmethodology, peptide display, or the like. Thus, the term antibody, asused herein, also includes antibody fragments either produced by themodification of whole antibodies or synthesized de novo usingrecombinant DNA methodologies. Antibodies also include single-armedcomposite monoclonal antibodies, single chain antibodies, includingsingle chain Fv(scFv) anti-bodies in which a variable heavy and avariable light chain are joined together (directly or through a peptidelinker) to form a continuous polypeptide, as well as diabodies,tribodies, and tetrabodies (see e.g. Pack et al. J Mol. Biol. 1995 Feb.10; 246(1):28-34; Pack et al. Biotechnology (N Y). 1993 November;11(11):1271-7; Pack & Plueckthun Biochemistry. 1992 Feb. 18;31(6):1579-84). The antibodies are e.g., polyclonal, monoclonal,chimeric, humanized, single chain, Fab fragments, single chain Fab (Hustet al., BMC Biotechnol (2007) 7:14), fragments produced by a Fabexpression library, or the like.

Polypeptides produced in accordance with the invention may be recoveredby methods known in the art. For example, the polypeptide may berecovered from the nutrient medium by conventional procedures including,but not limited to, centrifugation, filtration, ultrafiltration,extraction or precipitation. Purification may be performed by a varietyof procedures known in the art including, but not limited to,chromatography (e.g. ion exchange, affinity, hydrophobic,chromatofocusing, and size exclusion), electrophoretic procedures (e.g.,preparative isoelectric focusing), differential solubility (e.g.,ammonium sulfate precipitation) or extraction. Furthermore, thepolypeptide can be obtained from the host cells by cell disruption.

Also provided is a method for producing a vector nucleic acid asdescribed above comprising the step of assembling at least one cassette(Cas-POI), preferably an expression cassette (Exp-POI), into a vectorsuch that said cassette comprises a first polynucleotide (Pn-POI)encoding the polypeptide of interest, at least one stop codon downstreamof the first polynucleotide, and a second polynucleotide downstream ofthe stop codon encoding an immunoglobulin transmembrane anchor or afunctional variant. Said method may further comprise assembling

-   -   an expression cassette (Exp-MSM) comprising a mammalian        selectable marker gene,    -   an expression cassette (Exp-MASM) comprising a mammalian        amplifiable, selectable marker gene,        preferably such that the expression cassette (Exp-MASM) is        located 5′ and the expression cassette (Exp-MSM) is located 3′        of the expression cassette (Exp-POI) and wherein the expression        cassettes (Exp-MASM), (Exp-POI) and (Exp-MSM) are arranged in        the same 5′ to 3′ orientation.

Also provided is a eukaryotic, preferably a mammalian host cell which isobtained by the screening method described above. Also provided is aeukaryotic, preferably a mammalian host cell which comprises a cassette(Cas-POI) comprising a heterologous and hence foreign polynucleotideencoding a polypeptide of interest, at least one stop codon downstreamof said heterologous polynucleotide and a polynucleotide downstream ofthe stop codon encoding an immunoglobulin transmembrane anchor or afunctional variant thereof. The cassette (Cas-POI) may be introducede.g. by the vector nucleic acid according to the present invention.Preferably, the cassette (Cas-POI) and/or the cassette (Cas-POI′) is anexpression cassette.

Further features of the cassette (Cas-POI) and details of suitablevectors are described above and also apply to the host cell of thepresent invention. Suitable eukaryotic host cells are described above.Preferably, the eukaryotic host cell is a mammalian host cell. Accordingto one embodiment the eukaryotic host cell is not a B-cell or a B-cellderivative. Accordingly, the eukaryotic, preferably mammalian host cellis a host cell which does not naturally express the Ig alpha and Ig betareceptor chains. Furthermore, according to one embodiment, no artificialco-expression of the Ig alpha and Ig beta receptor chain occurs in saidhost cell. CHO cells are preferred host cells.

Also provided is a method for producing a eukaryotic host cell asdescribed above, wherein the eukaryotic host cell is transfected withthe vector nucleic acid according to the present invention and/or aheterologous nucleic acid comprising a cassette (Cas-POI) according tothe present invention. There are several appropriate methods known inthe prior art for introducing an expression vector into a mammalian hostcell. Respective methods include but are not limited to calciumphosphate transfection, electroporation, lipofection, biolistic- andpolymer-mediated genes transfer. Besides traditional random integrationbased methods also recombination mediated approaches can be used totransfer the cassette (Cas-POI) into the host cell genome. Suchrecombination methods may include use of site specific recombinases likeCre, Flp or ΦC31 (see e.g. Oumard et al, Cytotechnology (2006) 50:93-108) which can mediate directed insertion of transgenes.Alternatively, the mechanism of homologous recombination might be usedto insert the cassette (Cas-POI) (reviewed in Sorrell et al,Biotechnology Advances 23 (2005) 431-469). Recombination based geneinsertion allows to minimize the number of elements to be included inthe heterologous nucleic acid that is transferred/introduced to the hostcell. For example, an insertion locus might be used that alreadyprovides promoter and poly-A site (exogenous or endogenous) such thatonly the remaining elements (e.g. polynucleotide of interest, the stopcodon and polynucleotide encoding an immunoglobulin transmembrane anchorof functional fragment thereof) needs to be transferred/transfected tothe host cell. Even transfer of parts of the cassette (Cas-POI) would besufficient if the missing parts would be present at the insertion site.Embodiments of a suitable expression vector according to the presentinvention as well as suitable host cells and polypeptides of interestare described in detail above; we refer to the above disclosure.

Also provided is a polypeptide obtained by a method according to thepresent invention as defined above and in the claims. Said polypeptideis preferably an immunoglobulin molecule or a fragment thereof.Polypeptides produced according to the methods of the present inventiondepict good stability properties. The results also show that thepolypeptides are expressed in a functional form and hence in the rightconformation. Accordingly, the invention also provides polypeptidesobtained by the production method according to the present inventionusing the expression vector described in detail above. As is outlinedabove, polypeptides are obtained with a good yield due to theincorporated selection/screening step. The polypeptide is preferably animmunoglobulin molecule such as an antibody or a fragment thereof.

The invention is further illustrated by the following non-limitingexamples, which however, describe preferred embodiments of theinvention.

EXAMPLES Example 1 Vector Construction of the Ig Transmembrane Version

A synthetic 1113 bp DNA fragment encoding part of the IgG1 constantheavy chain region plus the leaky stop codon stuffer and the Igtransmembrane and cytoplasmic domain is inserted into pBW201 (a standardvector containing an IgG1 heavy chain and kappa light chain) via Age1and Asc1 generating pNT11 (see table 1). The nucleotide sequence of theIg transmembrane domain used is shown in SEQ ID No: 1, the leaky stopcodon stuffer is indicated. Of course, also variants of the encoded Igtransmembrane domain can be used according to the principles of thepresent invention which provide the same membrane anchoring function.Said variants are homologue to the encoded Ig transmembrane domain andcan e.g. be obtained by conservative amino acid substitution. They sharepreferably at least 80%, 85%, 90% homology. Polynucleotides encodingrespective variants e.g. hybridize to the shown sequence under stringentconditions.

The wt DHFR selection marker gene of pNT11 and pBW201 can be replaced bya synthetic 1252 bp fragment encoding a L23P point mutant of DHFR viaSwal and BgIII, thereby generating pNT29 and pBW478. The DHFR mutantallows the selection of dhfr+ cell lines.

The FACS vectors (pNT11, pNT29) are based on the standard vectors forantibody expression (pBW201, pBW478). pNT11 and pBW201 are differingfrom pNT29 and pBW478 in the DHFR selection marker cassette they arecarrying. Apart from that the backbones are identical. The vector has amono-cistronic “tandem” setup and contains antibody light and heavychain expression cassettes, both driven by the CMV promoter/enhancer.The only modification to generate the FACS vectors was the insertion ofan IgG1 transmembrane and cytoplasmic domain 3′ of the antibody heavychain (HC) cDNA. A short stuffer with a leaky translation terminationsignal is placed between HC and transmembrane domain. The sequenceenvironment selected for the stop codon is expected to lead to aread-through of up to 5%. All four vectors are coding for the same humanIgG antibody.

As is outlined above, the vector nucleic acids used for expression andin particular the orientation and arrangement of the vector elementschosen allow the very efficient expression of immunoglobulin molecules.Suitable vectors that can be used in conjunction with the presentinvention and which are described above are illustrated in the followingtable (the arrows indicate the 5′ to 3′ orientation of the geneticelements):

TABLE 1 Vector map pNT11 - “FACS vector” CMVprom/enhan → RK-intron →mAB-LC → SV40polyA → CMV prom/enhan → RK-intron → mAB-HC → Stuffer +leaky stop codon Ig transmembrane domain and cyto-plasmatic domain →SV40polyA → Phage f1 region → SV40prom/enhan → Neo → Synth polyA Amp →SV40prom/enhan → DHFR → SV40pA →

The abbreviations in table 1 have the regular meaning as apparent forthe person of skill in the art and as described above, and have inparticular the following meanings:

-   CMVprom/enh=human cytomegalovirus immediate early promoter/enhancer-   RK-intron=comprises the intron donor splice site of the CMV promoter    and the acceptor splice site of the mouse IgG Heavy chain variable    region (see e.g. Eaton et al., 1986, Biochemistry 25, 8343-8347,    Neuberger et al., 1983, EMBO J. 2(8), 1373-1378; it can be obtained    from the pRK-5 vector (BD PharMingen))-   mAB-LC=monoclonal antibody light chain-   mAB-HC=monoclonal antibody heavy chain-   SV40polyA=SV40 polyA site-   SV40prom/enhan=SV40 promotor/enhancer-   Neo=neomycin phosphotransferase-   Synth polyA=synthetic polyadenylation site-   Amp=beta lactamase antibiotic resistance gene-   DHFR=dihydrofolate reductase gene.

Example 2 Transfection and Selection of CHO-Cells

Cell cultivation, transfection and screening is carried out in shakeflasks using suspension growing CHO cells in a proprietary, chemicallydefined culture medium. Cells are either transfected by lipofection orelectroporation (nucleofection) following the manufacturer'sinstructions. Transfection efficiency is checked by transfecting a GFP(green fluorescence protein)-reporter plasmid and flow cytometricanalysis of the transfected cells. Depending on the cell viability,selection is started 24-48 h after transfection by adding G418containing selective medium to the cells. As soon as cells recover to aviability of above 80%, a second selection step is applied by passagingthe cells to G418 free, MTX (methotrexate) containing medium. Afterrecovery of the cells from the MTX selection, cultivation is continuedin MTX containing medium throughout FACS enrichment cycles, FACS cloningor limited dilution cloning and screening.

Cell viability and growth are monitored using an automated system(ViCell, Beckmann Coulter).

Example 3 FACS Analysis, Enrichment and Cloning of Cells

Labeling of cells: 2×10E7 cells per transfected pool are centrifuged andwashed with 5 mL of chilled PBS (phosphate buffered saline) andresuspended in 1 mL of cold PBS. A suitable amount of FITC (fluoresceinisothiocyanate) labeled anti-IgG antibody is added to the cells and isincubated on ice for 30 minutes in the dark. Subsequently, cells arewashed twice at room temperature with 5 mL PBS, resuspended in 1 mL PBS,filtrated and dispensed into a FACS tube for analysis, sorting andcloning.

Analysis, sorting and cloning of cells: The cell sorting is performedwith a FACSAria (Becton Dickinson) equipped with an Automatic CellDeposition Unit (ACDU) using FACSDiva software. A low powered air-cooledand solid-state laser (Coherent® Sapphire™ solide state) tuned to 488 nmis used to excite fluorescein dyes bound to the secondary antibody. Therelative FITC fluorescence intensity is measured on E detector through a530/30 BP filter. Five percents of the highest FITC fluorescent cellsare gated and sorted either in block or as single cells in 96 wellplates.

Example 4 Determination of Clonal Productivity and Stability

Productivity of clones is analyzed in batch and fed batch experimentsusing different formats. Initial clone screening is performed in 24-wellplate batch assays by seeding cells to shaken 24-well plates. Antibodyconcentrations in the cell culture supernatant are determined byprotein-A HPLC 10d after starting the culture. The highest producingclones are also analyzed in shake flask models in batch and fed batchmode. Batch cultures are seeded in shake flask 500 with 100 mL workingvolume and are cultivated in a shaker cabinet (not humidified) at 150rpm and 10% CO₂. Viability of cells should be >90% when starting theassay. The seeding cell density is 2×10⁵ c/mL. Productconcentration/cell number/viability determination took place at day 3-7,10 and 13. Fed batch experiments are done using the same conditions butwith a starting cell density of 4×10⁵ c/mL and with regular adding offeeds. Clonal stability is evaluated by culturing the cells over aperiod of 14 weeks with productivity measurements using the shake flaskbatch model every two weeks.

Example 5 Analysis of Transiently Transfected Cells

To test whether membrane bound translation products are present on thecell surface after transfection with the new FACS vector (here pNT11 orpNT29), transiently transfected cells are analyzed by immunostaining andflow cytometry. 48 h after transfection, cells are stained with aFITC-labeled antibody directed against human IgG. Cells transfected witha GFP expression vector are used as a transfection control, thetransfection efficiency is calculated to be about 60%. Un-transfectedcells and cells transfected with the standard vector (not comprising atransmembrane domain) do not show significant levels of surfaceassociated antibody, while 16% of the cells transfected with the FAGSvector are stained above background level. This shows that the fusionpeptide, here an antibody molecule, anchored to the cell membrane can bedetected on the cell surface.

Example 6 Analysis and Enrichment of Stable Transfected Cells

Having shown presence of membrane bound antibody on transientlytransfected cells the surface expression level and distribution inselected pools of transfected cells is analysed. Thereby, it can beshown that producing cells can be selectively enriched by FACS sorting.Therefore, cells after transfection are selected with G418 andsubsequently with MTX. The resulting pools of resistant cells arestained with FITC labelled anti-IgG antibody and analyzed byflow-cytometry. As a control, un-transfected cells are stained andanalyzed. Subpopulations of positive cells are detected in the selectedpools transfected with the FACS vector. The distribution of positivecells thereby differed between the two analyzed pools. To assess whetherhigh producing cells can be enriched based on their fluorescence signal(and hence allow a quantitative selection), cells having the highestfluorescence intensity are sorted (top 5%) from each of the two poolsand sub-cultured to compare the productivity with the pool beforeenrichment.

Example 7 Analysis of Productivity of Enriched and Non Enriched Cells

Productivity analyses of the selected pools before and afterflow-cytometry enrichment are done in shake flask batch cultures tocompare the end-product concentration at day 13. At day 13 thesupernatant is harvested and analyzed for IgG content by Protein-A-HPLC.Both pools show a significant increase of production level already afterperforming one FACS enrichment cycle according to the teachings of thepresent invention. While product concentration for pool 1 increases by afactor of approximately 2, pool 2 increases by a factor of almost 10showing that high producing cells are selectively detected duringstaining and sorting.

Already in the first enrichment cycle antibody concentrations of almost250 mg/l can be obtained.

Example 8 Flow-Cytrometry Based Selective Cloning of High ProducingCells

Flow-cytrometry can be used to sort and seed individual stained cellsaccording to their staining profile. To analyze whether such selectivecloning results in higher number of high producing clones than cloningby limiting dilution, clones are generated using both methods andproductivity is analyzed in 24-well plate batch cultures. Batch culturesin 24-well plates are done and at day 10 supernatants are harvested andmeasured for IgG content by Protein-A-HPLC. The results are as follows:

TABLE 2 FACS sorting versus limited dilution (LD) 0-25 26-50 51-7576-100 101-125 126-150 Method mg/l mg/l mg/l mg/l mg/l mg/l LD -obtained 12 0 1 0 1 0 clones FACS - obtained 2 2 2 2 0 1 clones

The flow-cytrometry derived clones have a higher average productivitycompared to the liming dilution derived clones, which is also reflectedin the clonal distribution of the productivity range.

Example 9 Comparison of FACS and Standard Vector

To confirm the beneficial effect of flow-cytometry enrichment oftransfected cells and to compare use of the FACS vector (pNT29) with astandard vector, cells are transfected and selected with G418 and MTX.Three cell pools transfected with the FACS vector (samples 1, 2 and 3)and three cell pools transfected with the standard vector (samples 7, 9and 9) are analyzed by flow cytrometry and the 5% having the higheststaining signal are sorted. Shake flask batch cultures are done tocompare the increase of product concentration after enrichment.Transfected and selected pools are stained and sorted by flow-cytrometryto enrich the top 5% based on the fluorescence intensity. Before andafter enrichment, shake flask batch cultures are done and after 13 dayssupernatants are analyzed by Protein-A-HPLC. The results are as follows(approximately):

TABLE 3 Results obtained with the FACS vector Sample Productconcentration Sample 1; 10 mg/l   FACS vector, before enrichment Sample1; 55 mg/ml FACS vector, 1st enrichment Sample 1; 100 mg/ml  FACS 2ndenrichment Sample 1; 365 mg/ml  FACS vector, 3rd enrichment Sample 2; 40mg/ml FACS vector, before enrichment Sample 2; 65 mg/ml FACS vector, 1stenrichment Sample 2; 90 mg/ml FACS vector, 2nd enrichment Sample 2; 340mg/ml  FACS vector, 3rd enrichment Sample 3; 15 mg/ml FACS vector,before enrichment Sample 3; 95 mg/ml FACS vector, 1st enrichment Sample3; 155 mg/ml  FACS vector, 2nd enrichment Sample 3; 85 mg/ml FACSvector, 3rd enrichment

TABLE 4 Results obtained with the Standard vector Sample Productconcentration Sample 7; 40 mg/l   Standard vector, before enrichmentSample 7; 55 mg/ml Standard vector, 1st enrichment Sample 7; 50 mg/mlStandard 2nd enrichment Sample 7; 25 mg/ml Standard vector, 3rdenrichment Sample 8;  5 mg/ml Standard vector, before enrichment Sample8; 10 mg/ml Standard vector, 1st enrichment Sample 8; 12 mg/ml Standardvector, 2nd enrichment Sample 8; 15 mg/ml Standard vector, 3rdenrichment Sample 9;  5 mg/ml Standard vector, before enrichment Sample9;  2 mg/ml Standard vector, 1st enrichment Sample 9; 10 mg/ml Standardvector, 2nd enrichment Sample 9; 10 mg/ml Standard vector, 3rdenrichment

As is demonstrated by the results, the production level of FACS vectortransfected cells increases significantly for the tested three pools,while in case of the standard vector only one pool showed a significantincrease in product concentration. The average of product concentrationsafter enrichment with the FACS vector is significantly higher as withthe standard vector. Two further sequential FACS enrichment cycles aredone to enrich high producing cells showing that only in case of theFACS vector productivity of the cell populations is increased. Finally,product concentrations can be increased by 4- to 30-fold.

For comparison of the suitability of both vectors for selective cloning,clones from non-enriched pools with comparable productivity areselectively sorted by flow-cytometry. Subsequently, productivity of theclones is analyzed in 24-well batch cultures. Clones derived from FACSvector transfected pools are found to have a higher average expressionlevel as clones from standard vector transfected pools. The clonaldistribution of productivity shows that in case of the FACS vector ahigher number of good producing clones is obtained (see table 5):

TABLE 5 Standard vector versus FACS vector (pNT29) 0-50 51-100 101-150151-200 201-250 251-300 301-350 Method mg/l mg/l mg/l mg/l mg/l mg/lmg/ml Stan- 31 7 0 2 0 1 0 dard vector FACS 21 20 4 4 4 2 1 vector

Example 10 Further Comparisons Between the FACS Vector and StandardExpression Vectors a) Vector Construction

The vectors pBW201, pNT11, pBW478 and pNT29 are obtained as described inexample 1.

b) Transfection, Selection and Cloning of CHO Cells

This is done as described in example 2.

c) FACS Analysis, Enrichment and Cloning of Cells

This is done as described in example 3.

d) Determination of Antibody Production and Clonal Stability

The productivity of clones and pools is analyzed in batch and fed batchexperiments using different formats. Pools before and after FACSenrichment are analyzed in shake flask batch assays by seeding 1×10⁵cells per mL (c/mL) in 50 mL working volume using shake flasks with 250mL capacity. IgG content is analyzed by Protein-A HPLC from samplestaken at day 13 of the batch culture. Initial screening of clones isperformed in 24-well plate batch assays by seeding cells into shaken24-well plates. Antibody concentrations in the cell culture supernatantare determined by quantitative Protein A-HPLC 10 days after starting theculture. The highest producing clones are analyzed in shake flask modelsin batch and fed batch mode. Batch cultures are seeded into shake flasks(500 mL capacity) with 100 mL working volume and are cultivated in ashaker cabinet (not humidified) at 150 rpm, 36.5° C. and 10% CO₂.Viability of cells is >90% when starting the assay. The seeding celldensity is 2×10⁵ c/mL. Antibody concentrations, cell number andviability are determined on days 3-7, 10 and 13. Fed batch experimentsare done using the same conditions but with a runtime of 17 days andwith a starting cell density of 4×10⁵ c/mL and with regular addition offeeds starting at viable cell densities above 7×10⁶ c/mL. Clonalstability is evaluated by culturing the cells over a period of 12 weekswith productivity measurements using the shake flask batch model everytwo weeks.

e) Analysis and Enrichment of Stable Transfected Cells

The surface expression in stably transfected cell populations isanalysed to test whether producing cells can be selectively enriched byFACS-sorting. Therefore, cells after transfection are selected with G418and subsequently with MTX. The resulting pools (10 per vector) ofresistant cells are stained as described above and analyzed byflow-cytometry. With the used staining protocol positive sub-populationsof cells could be detected in both, pBW478 and pNT29 transfected cellpools. As expected, a higher proportion of FACS positive cells is foundwith the FACS vector.

To show that high producing cells can be enriched based on theirfluorescence signal, cells having the highest fluorescence intensity aresorted (top 5%) from the individual cell pools and sub-cultured tocompare the productivity with the pool before enrichment. A second cycleof enrichment is performed after expansion and pooling of the one timessorted cell populations. The percentage of staining positive cellssurprisingly increased most with the standard vector in the firstenrichment cycle. FACS-vector transfected pools showed similarenrichment factors with the used staining protocol and generally,significant pool to pool variation was observed. After the secondenrichment cycle, almost homogeneously FACS positive cell populationsare obtained (see Table 6a and 6b).

Table 6a and 6B: Average Staining Results and Productivities Before andafter Sorting

TABLE 6a FACS analysis of stained cells before and after FACS enrichmentcycles % cells above background pBW478 (reference vector) pNT29 (FACSvector) FITC staining No FACS 1x FACS 2x FACS No FACS 1x FACS 2x FACSAVG 5.9 83.2 90.4 14.2 46.6 90.5 STDD 3.396403 15.80158 1.1267958.974284 13.51148 1.422439 Table 6a: Transfected and selected cell poolswere stained for surface IgG. Average percentage of cells stained abovethe level of untransfected cells is shown. Before enrichment a higherpercentage of positive cells is found with the FACS vector. After thefirst enrichment cycle of the top 5%, the proportion of stainingpositive cells was highest with the standard vector. After the secondenrichment cycle, greater that 90% of all cells were positive with bothapproaches. Abbreviations: AVG: Average and STDD: Standard deviation.

TABLE 6b Productivities of in shake flask batch model before and afterFACS enrichment cycles mAb pBW478 (reference vector) pNT29 (FACS vector)(mg/L) No FACS 1x FACS 2x FACS No FACS 1x FACS 2x FACS AVG 38.5 123.468.3 46.5 171.8 363 STDD 17.66509 101.8563 6.592926 15.30614 114.193670.19259 Table 6b: Productivity of cell pools is analyzed from shakeflask batch cultures by Protein-A HPLC at day 13 of the culture. Thefirst enrichment cycle led to a significant increase of productivity inboth cases. After the second enrichment, only FACS vector transfectedcell pools showed additional increase in productivity.

f) Analysis of Productivity of Enriched and Non Enriched Cells

Productivity analysis of the selected pools before and afterflow-cytometry enrichment are done in shake flask batch cultures tocompare the end-titers at day 13. Productivity of the pools beforeenrichment is in a very comparable range for both vectors used. With thefirst enrichment cycle on the individual pools significant improvementof the average productivity is achieved with all approaches and again,there is substantial variation between individual pools (see Table 6b).Surprisingly, the productivity of the standard vector transfected poolsis not higher compared to the FACS vector transfected ones although amuch higher level of FACS staining positive cells was seen before. Bysorting a second time from the pooled sorted cell populations, nofurther improvement of productivity is achieved with the standardvector. In contrast, a lower productivity is obtained although theFACS-staining result suggested that almost 100% of the cells should beproducing antibody (see Table 6a). Productivity of FACS-vectortransfected pools could be significantly improved by sorting a secondtime. The used FACS procedure leads to more selective enrichment of highproducing cells with a productivity increase of about at least 8-foldcompared to unsorted population and at least 2-fold compared to thepools after one sorting.

g) Flow-Cytrometry Based Selective Cloning of High Producing Cells

Flow-cytrometry can be used to sort and seed individual stained cellsaccording to their staining profile. To analyze whether such selectivecloning results in higher number of high producing clones when using theFACS-vector compared to the standard vector, clones are generated usingboth methods and productivity is analyzed in 24-well plate batchcultures.

In a first round, cells are directly FACS-cloned from the MTX selectedcell pools without any pre-enrichment step. Three pools per vector arechosen based on their staining profile. Clones are generated from thetop 5% of the stained cell pools and in total about 500 clones areanalyzed. While average productivity of clones with the reference vectorwas 39 mg/L, FACS-vector clones produced an average of 87 mg/L. As shownin Table 7a, this is also reflected by the clonal distribution whichconfirms that a much higher proportion of high producing clones isobtained with the FACS-vector. Interestingly, one out of the over 270clones analyzed from the standard vector transfection had an almost2-fold higher productivity compared to the others. This exceptionalclone is designated LP. Identifying such a high producing cell clonewith the standard vector setup in combination with a FACS screeningprocedure is thus generally possible. However, it is a very rare andthus lucky event. This is also the decisive difference to the selectionprocess according to the teachings of the present invention. While thestandard set up allows the selection of (very) high producers only inexceptional and thus rare cases, the method according to the presentinvention allows the selection of (very) high producers reproducibly andthus reliably.

A second FACS-cloning experiment is performed starting from the 10pooled populations per vector after the first enrichment cycle. Thistime approximately 240 clones are screened in 24-well batch cultures.Again, clones obtained with the FACS-vector have a much higher averageproductivity than the reference standard vector. No improvement comparedto cloning without pre-enrichment is achieved with the reference vectorat an average clone productivity of 40 mg/L. The LP clone was notidentified again. In case of the FACS vector transfected clones, anaverage productivity of 275 mg/L was obtained with the used FACS method.The clonal distribution clearly demonstrates the superiority of theFACS-vector setup with regards to selective cloning of high producers(see Table 7b).

Table 7a and 7b: Comparison of Productivity of Clones

TABLE 7a 24-well-Batch - Clonal distribution pBW478 pNT29   0-50 mg/L196 163  51-100 mg/L 70 22 101-150 mg/L 8 11 151-200 mg/L 2 5 201-250mg/L 0 13 251-300 mg/L 2 9 301-350 mg/L 1 10 351-400 mg/L 0 6 401-450mg/L 0 5 451-500 mg/L 0 2 501-550 mg/L 0 1 551-600 mg/L 1 0 Table 7a:Clones are generated by flow-cytrometry from the top 5% of the threestained cell pools with the highest percentage of staining positivecells after selection. For productivity assessment, batch cultures in24-well plates were done and at day 10 supernatants were harvested andmeasured for IgG content by Protein-A-HPLC. Shown here is the clonaldistribution of the productivity range. A significantly higherproportion of high producing clones is obtained when using the FACSvector (pNT29).

TABLE 7b 24-well-Batch: FACS Cloning Pooled pools pBW478 pNT29   0-50mg/L 102 22  51-100 mg/L 17 2 101-150 mg/L 13 5 151-200 mg/L 5 3 201-250mg/L 2 7 251-300 mg/L 0 11 301-350 mg/L 0 11 351-400 mg/L 0 15 401-450mg/L 0 9 451-500 mg/L 0 7 501-550 mg/L 0 7 551-600 mg/L 0 1 601-650 mg/L0 3 Table 7b: Clones obtained by FACS cloning from the top 5% of stainedcombined pools after one enrichment cycle were analyzed. No benefit thefrom pre-enrichment was found for reference vector (pBW478) transfectedcells, while in case of FACS vector (pNT29) transfected cells,pre-enrichment led to a significant reduction of non-producers and to anincrease of the average productivity of clones.

h) Characterisation of Clones

The LP clone derived from the standard vector as well as 10 highproducing FACS-vector clones are expanded to shake flasks and tested ingeneric shake flask batch and fed-batch models to evaluate theirmanufacturing potential.

Productivity in batch cultures is found to be about the same in therange of 1 g/L for all clones tested. Fed-batch productivities are alsovery comparable for all clones and in the range of 3.5-4 g/L (see Table8). No significant difference in growth parameters are observed whencomparing the FACS-vector transfected clones with reference vectortransfected clones (LP and clones from previous experiments). Also,production stability is found to be high for the FACS-vector derivedclones, only one out of 10 analyzed clones showed a drop of productivitygreater than 25% after 12 weeks in culture which is a lower ratio ofunstable clones as it was observed with the reference standard vector inprevious experiments (data not shown).

TABLE 8 Pool productivities: Fed batch shake flask (SF) model mAb (g/L)SF batch SF fed batch 1 = LP 1.1 4.3 2 0.9 3.7 3 1.0 3.9 4 1.0 3.8 5 1.03.8 6 1.0 3.9 7 1.2 3.3 8 1.0 3.8 9 0.9 3.6 10  1.0 3.7 11  0.9 3.3Table 8: The highest producing clone obtained with the standard vector(pBW478) and 10 clones derived from the FACS vector (pNT29) are analyzedin batch and fed batch shake flask cultures. IgG content is analyzed byProtein-A HPLC at day 13 (batch cultures) or day 17 (fed batchcultures). All analyzed cones produce in a comparable range.

Example 11 Large Scale Production of Polypeptides with Transfected CHOCells

The production of polypeptides in large scale can be done for example inwave, glass or stainless steel bioreactors. For that purpose the cellsare expanded, usually starting from a single frozen vial, for example avial from a Master Cell Bank. The cells are thawed and expanded throughseveral steps. Bioreactors of different scale are inoculated withappropriate amounts of cells. The cell density can be increased byadding feed solutions and additives to the bioreactor. Cells are kept ata high viability for a prolonged time. Product concentrations in thereactor ranging from a few hundred milligrams per litre up to severalgrams per litre are achieved in the large scale. Purification can bedone by standard chromatography methodology, which can include affinity,ion exchange, hydrophobic interaction or size exclusion chromatographysteps. The size of the bioreactor can be up to several thousand litresvolume in the final scale (see also e.g. F. Wurm, Nature BiotechnologyVol. 22, 11, 2004, 1393-1398).

1. A method for selecting at least one eukaryotic host cell expressing adesired level of a polypeptide of interest, comprising: a) providing aplurality of eukaryotic host cells comprising a heterologous nucleicacid comprising at least one cassette (Cas-POI) comprising at least afirst polynucleotide (Pn-POI) encoding the polypeptide of interest, atleast one stop codon downstream of the first polynucleotide, and asecond polynucleotide downstream of the stop codon encoding animmunoglobulin transmembrane anchor or a functional variant thereof; b)cultivating the eukaryotic host cells to allow expression of thepolypeptide of interest such that at least a portion of the polypeptideof interest is expressed as a fusion polypeptide comprising theimmunoglobulin transmembrane anchor or a functional variant thereof,wherein said fusion polypeptide is being displayed on the surface ofsaid host cell; c) selecting at least one eukaryotic host cell basedupon the presence or amount of the fusion polypeptide displayed on thecell surface.
 2. A method for producing a polypeptide of interest withhigh yield, the method comprising: a) providing a plurality ofeukaryotic host cells comprising a heterologous nucleic acid comprisingat least one cassette (Cas-POI) comprising a first polynucleotide(Pn-POI) encoding the polypeptide of interest, at least one stop codondownstream of the first polynucleotide, and a second polynucleotidedownstream of the stop codon encoding an immunoglobulin transmembraneanchor or a functional variant thereof; b) cultivating the eukaryotichost cells to allow expression of the polypeptide of interest such thatat least a portion of the polypeptide of interest is expressed as afusion polypeptide comprising the immunoglobulin transmembrane anchor ora functional variant thereof, wherein said fusion polypeptide is beingdisplayed on the surface of said host cell; c) selecting at least oneeukaryotic host cell based upon to the presence or amount of the fusionpolypeptide displayed on the cell surface; d) culturing the selectedeukaryotic host cell in culture medium under conditions that allow forexpression of the polypeptide of interest.
 3. The method according toclaim 1, wherein expression of the cassette (Cas-POI) results in atranscript comprising at least a first polynucleotide, whereintranslation of said first polynucleotide results in the polypeptide ofinterest; at least one stop codon downstream of said firstpolynucleotide; a second polynucleotide downstream of said stop codon,wherein translation of said second polynucleotide results in theimmunoglobulin transmembrane anchor or a functional variant thereof,wherein at least a portion of the transcript is translated into a fusionpolypeptide comprising the immunoglobulin transmembrane anchor or afunctional variant thereof by translational read-through of the at leastone stop codon.
 4. The method according to claim 1, wherein theimmunoglobulin transmembrane anchor is selected from the groupconsisting of a) a transmembrane anchor derived from IgA, IgE, IgM, IgGand/or IgD, b) an immunoglobulin transmembrane anchor comprising acytoplasmatic domain, c) an immunoglobulin transmembrane anchorcomprising a sequence as shown in SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO:4, SEQ ID NO: 5, SEQ ID NO: 6 and/or SEQ ID NO: 7 and d) functionalvariants of the foregoing, which allow anchoring of the fusionpolypeptide to the surface of the eukaryotic host cell.
 5. The methodaccording to claim 1, wherein step c) comprises contacting the pluralityof eukaryotic host cells with a detection compound binding the fusionpolypeptide and selecting at least one eukaryotic host cell based uponthe presence or amount of the bound detection compound.
 6. The methodaccording to claim 3 wherein stop codon read-through results inapproximately ≦50%, ≦25%, ≦15%, ≦10%, ≦5%, ≦2.5%, ≦1.5%, ≦1% or lessthan ≦0.5% of fusion polypeptide.
 7. The method according to claim 1,wherein two or more selection cycles are performed, wherein in eachselection cycle at least one eukaryotic host cell is selected based uponthe presence or amount of the fusion polypeptide displayed on the cellsurface.
 8. The method according to claim 5, wherein binding of thedetection compound to the surface of the eukaryotic host cell isdetected by flow cytometry. 9-14. (canceled)
 15. The method according toclaim 2, wherein expression of the cassette (Cas-POI) results in atranscript comprising at least a first polynucleotide, whereintranslation of said first polynucleotide results in the polypeptide ofinterest; at least one stop codon downstream of said firstpolynucleotide; a second polynucleotide downstream of said stop codon,wherein translation of said second polynucleotide results in theimmunoglobulin transmembrane anchor or a functional variant thereof,wherein at least a portion of the transcript is translated into a fusionpolypeptide comprising the immunoglobulin transmembrane anchor or afunctional variant thereof by translational read-through of the at leastone stop codon.
 16. The method according to claim 2, wherein theimmunoglobulin transmembrane anchor is selected from the groupconsisting of a) a transmembrane anchor derived from IgA, IgE, IgM, IgGand/or IgD, b) an immunoglobulin transmembrane anchor comprising acytoplasmatic domain, c) an immunoglobulin transmembrane anchorcomprising a sequence as shown in SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO:4, SEQ ID NO: 5, SEQ ID NO: 6 and/or SEQ ID NO: 7 and d) functionalvariants of the foregoing, which allow anchoring of the fusionpolypeptide to the surface of the eukaryotic host cell.
 17. The methodaccording to claim 2, wherein step c) comprises contacting the pluralityof eukaryotic host cells with a detection compound binding the fusionpolypeptide and selecting at least one eukaryotic host cell based uponthe presence or amount of the bound detection compound.
 18. The methodaccording to claim 15, wherein stop codon read-through results inapproximately ≦50%, ≦25%, ≦15%, ≦10%, ≦5%, ≦2.5%, ≦1.5%, ≦1% or lessthan ≦0.5% of fusion polypeptide.
 19. The method according to claim 2,wherein two or more selection cycles are performed, wherein in eachselection cycle at least one eukaryotic host cell is selected based uponthe presence or amount of the fusion polypeptide displayed on the cellsurface.
 20. The method according to claim 17, wherein binding of thedetection compound to the surface of the eukaryotic host cell isdetected by flow cytometry.
 21. A vector nucleic acid suitable forexpressing at least one polypeptide of interest in a eukaryotic hostcell, comprising a) at least one cassette (Cas-POI) comprising aninsertion site for a first polynucleotide (Pn-POI) encoding thepolypeptide of interest and/or a first polynucleotide encoding apolypeptide of interest, b) at least one stop codon downstream of saidinsertion site and/or downstream of the first polynucleotide, and c) asecond polynucleotide downstream of the stop codon encoding animmunoglobulin transmembrane anchor or a functional variant thereof. 22.The vector nucleic acid according to claim 21, comprising at least oneof the following characteristics: a first polynucleotide (Pn-POI)encoding the polypeptide of interest in the cassette (Cas-POI); anexpression cassette (MSM) comprising a mammalian selectable marker gene;and/or an expression cassette (MASM) comprising a mammalian amplifiable,selectable marker gene.
 23. The vector nucleic acid according to claim21 for expressing at least one immunoglobulin molecule or a functionalvariant thereof, comprising an expression cassette (Exp-POI) comprisinga first polynucleotide encoding the heavy chain of an immunoglobulinmolecule or a functional fragment thereof, at least one stop codondownstream of the first polynucleotide, and a second polynucleotidedownstream of the stop codon encoding an immunoglobulin transmembraneanchor or a functional variant thereof; and an additional expressioncassette (Exp-POI′) comprising a polynucleotide encoding thecorresponding light chain of an immunoglobulin molecule or a functionalfragment thereof.
 24. A method for producing a vector nucleic acidaccording to claim 21, wherein the method comprises assembling at leastone cassette (Cas-POI) into a vector such that said cassette (Cas-POI)comprises a first polynucleotide (Pn-POI) encoding the polypeptide ofinterest, at least one stop codon downstream of the firstpolynucleotide, and a second polynucleotide downstream of the stop codonencoding an immunoglobulin transmembrane anchor or a functional variantthereof.
 25. A eukaryotic host cell comprising a cassette (Cas-POI)comprising at least a heterologous polynucleotide encoding apolynucleotide of interest, at least one stop codon downstream of saidheterologous polynucleotide and a polynucleotide downstream of the stopcodon encoding an immunoglobulin transmembrane anchor or a functionalvariant thereof; wherein said eukaryotic host cell is optionallyobtained by the method according to claim
 1. 26. A eukaryotic host cellcomprising a cassette (Cas-POI) comprising at least a heterologouspolynucleotide encoding a polynucleotide of interest, at least one stopcodon downstream of said heterologous polynucleotide and apolynucleotide downstream of the stop codon encoding an immunoglobulintransmembrane anchor or a functional variant thereof; wherein saideukaryotic host cell comprises a vector nucleic acid according to claim21.
 27. A method for producing a polypeptide of interest, said methodcomprising culturing a eukaryotic host cell according to claim
 25. 28. Amethod for producing a polypeptide of interest, wherein a eukaryotichost cell according to claim 26 is cultured for expressing thepolypeptide of interest.
 29. The method for producing a polypeptide ofinterest according to claim 2, further comprising at least one stepselected from the steps: obtaining the polypeptide from the cellculture; obtaining the polypeptide from the culture medium wherein thepolypeptide is secreted into the culture medium; disrupting theeukaryotic host cells to obtain the expressed polypeptide; isolating theexpressed polypeptide; purifying the expressed polypeptide; and furtherprocessing or modifying the expressed polypeptide.
 30. The method forproducing a polypeptide of interest according to claim 27, furthercomprising at least one step selected from the steps: obtaining thepolypeptide from the cell culture; obtaining the polypeptide from theculture medium wherein the polypeptide is secreted into the culturemedium; disrupting the eukaryotic host cells to obtain the expressedpolypeptide; isolating the expressed polypeptide; purifying theexpressed polypeptide; and further processing or modifying the expressedpolypeptide.